USA; NBA TV sideline reporter Dennis Scott (left) interviews Los Angeles Lakers forward LeBron James after the game against the Portland Trail Blazers at Crypto.com Arena Mandatory Credit: Kirby Lee-USA TODAY Sports As the buzzer sounded on a Boston Celtic blowout of the Orlando Magic Tuesday night it marked the end of an era for the NBA on television as the final playoff game on NBA TV Yet it remains to be seen whether it will be the league-owned network’s final game overall Little is known about what NBA TV will look like next season. As part of a settlement the sides reached last year, the NBA will pay Warner Bros. Discovery $70 million/year over five years to continue producing content for the league’s various platforms — including NBA TV Will that include WBD continuing to produce studio programming Could that even include continued production of live games on NBA TV assuming there are any live games on NBA TV To say details are scarce would be an understatement What is a virtual certainty is that any future version of NBA TV will play a far smaller role in the league’s television strategy With so much NBA inventory spoken for next season — Mondays on Peacock Sundays on NBC and Peacock — any live game presence on NBA TV will surely be a far cry from its current 96-game regular season schedule Maybe a few Thursday night and weekend games during the football season before Amazon and the broadcast networks ramp up their schedules For a network that has carried games — both regular season and postseason — for more than 22 years Yet that is the broader trend for league-owned networks three-year-old NFL Network became the exclusive home of Thursday Night Football debuting in the plum territory of Thanksgiving night While its original schedule was limited to eight late season games the network would expand to a 13-week schedule in 2012 that spanned virtually the entire season from Week 2 to Week 15 (not including Thanksgiving) When the NFL finally began selling rights to TNF in 2014 NFL Network continued to air all games — simulcasting the games that aired on CBS (and later NBC and FOX) and carrying a handful of exclusive windows After nearly a decade of broadcast networks paying hundreds of millions for non-exclusive rights when the NFL sold TNF rights to Amazon as part of the 2021 media rights deal (for double what the broadcast networks were paying) it was a fully exclusive deal but it is a long way from owning exclusive rights to a primetime package it carries most of the 9:30 AM ET International Series games from Europe plus a late season double or tripleheader here and there NFL Network used to get holiday games on Thanksgiving and Christmas — including a game on the latter just four years ago — but the chances of the league ever again setting aside valuable holiday inventory for its in-house platform are next to nothing The NFL has been trying to offload its media apparatus for years off-again negotiations to cede control to ESPN which are both operated by MLB Advanced Media but they too have lost key inventory in recent years The days of MLB Network airing two Division Series games per year ended with the start of the current MLB TV deal in 2022 So have the days of NHL Network getting an occasional Stanley Cup playoff game also owing to a new media rights deal in 2022 Even the venerable Golf Channel is faces a murky future as one of the several NBC Universal networks being spun off into the new venture “SpinCo.” Tennis Channel which this week hired Amazon executive Jeff Blackburn as CEO has long been the subject of sale rumors and as of this year no longer has its lone Grand Slam While not analogous to league-owned networks — both channels have always existed independently of the tours they cover — the uncertainty is another indication that sport-specific channels may not be long for the television world Some have already gone by the wayside. Speed Channel and Fox Soccer long ago transformed into FS1 and FXX, respectively. ESPN last year shut down the University of Texas Longhorn Network, which has since relaunched as a FAST channel the complete collapse of the conference being the culprit It is simply the case that setting aside big games for league-owned networks no longer makes sense in an era of shrinking cable subscriptions and rising media rights revenues Why would leagues reserve quality live game content for their own declining networks when any number of third-party platforms would be willing to pay exorbitant sums for those rights That there was ever a time when playoff games aired on NBA TV and MLB Network a primetime NFL package belonged exclusively to NFL Network majors aired on Golf Channel and Tennis Channel and big football games aired on Longhorn Network and Pac-12 Network may be hard to believe in a decade or so — maybe even sooner all-encompassing coverage for dedicated fans What they can no longer do is compete with the rights-paying partners who pay the bills John Skipper is leaving Meadowlark Media; Fubo still planning to launch a sports "skinny bundle"; the future of the.. The NFL Draft averaged its largest audience on record save for the anomalous COVID edition five years ago A thriller between the Lakers and Timberwolves scored the third-largest opening round NBA playoff audience since the current media.. the second night of the NFL Draft surged by 40 percent.. “any number of third-party platforms would be willing to pay exorbitant sums for those rights” From what I’m reading,.MLB isn’t finding thise exorbitant sums my understanding is that the crumbling of the television bundle is making these mid-tier specialty channels to lose bargaining power I know obviously there will be a new NBA tv contract starting next year I figure they will probably show some early season games also since NBA TV is owned by TNT will they still have their pregame and postgame hosts If you’re talking about ‘Inside the NBA,’ no chance Charles Barkley would leave the $1000-a-hand Blackjack table with the dealer showing 12 dial up Zaslav and yell for 30 straight minutes if he and the rest still had to do NBATV with weekends hemmed up by ABC and a ‘general sports interest’ TNT version that will almost certainly last for less time than Charles’s show with Gayle King I had not previously considered how many niche sports channels have existed I watch much more college than “big 4” professional sports BTN and SECN seem bigger than ever for fans of their conferences I suppose eventually all SECN/BTN content could flow elsewhere perhaps ESPN+/OTT for SECN and FOX’ planned DTC offering for BTN there is so much inventory for these conferences – much of it medium or occasionally even high demand – that BTN and SECN still seem essential I think MLB and NHL networks are more important to their fans than NFL and NBA The amount of content that ESPN gives to the NFL and NBA make those channels obsolete The fans know they will get the information/highlights they need from ESPN NHL content is improving since ESPN got the rights back I can’t see ESPN investing in MLB content Can’t see any reason MLB network will stop existing A few months ago MLB Network announced that they would build a new larger studio operation a few miles away from where they are now Presumably this is to accommodate the extra production for their centralized streaming concept So they are betting on some kind of long-term viability maybe with the current network morphing into a subchannel of MLB.TV But I haven’t seen anything about what will happen to the NHL Network by then Sorry that it’s niche-level specific but I could see NBA TV pivot to some kind of Hybrid-FAST model like Reelz uses for the show On Patrol: Live (aka COPS Red Zone) Most of the time they’re showing classic episodes and other cheap-to-produce ancillary content then it’s show time on Friday and Saturday nights when they’re actually live Media partners and RSNs would have to play along but I could see NBA TV showing classic games all day until 7p rolls around and they either show the 2 best locally-produced games of the night or they produce a nightly Red Zone style show with the locally-produced games it could be a way for the league to try to retain eyeballs nightly I also think NBA TV could one day end up as a FAST channel but they’ll be limited even on whiparound rights I think ESPN has exclusive rights to that style of programming in the new deal Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" Learn how to describe the purpose of the image (opens in a new tab) Leave empty if the image is purely decorative Learn how to describe the purpose of the image (opens in a new tab). Leave empty if the image is purely decorative. Start your free trial with Shopify today—then use these resources to guide you through every step of the process A niche market is a specialized segment within a broader market Here are 11 niche markets and relevant products to explore On this pageWhat is a niche market?11 niche market examples How to find niche markets How to evaluate your niche market ideas Creating a unique selling point (USP)Niche market FAQStart your online business today. Ever heard the saying “Jack of all trades, master of none?” While a general store can cater to a broad range of customers, a niche business delivers best-in-class service to a specific target audience. you can pinpoint what customers truly need and build a trusted brand that solves unique challenges If you’re struggling to find a product idea for your business, identifying a niche is a great starting point. This post explains what a niche market is, offers 11 niche market examples, and outlines how to discover relevant, in-demand products A niche market is a specific group of consumers with shared characteristics and preferences that make them more inclined to buy specific products or services well-defined groups within target markets and often demonstrate a higher willingness to pay for solutions tailored to their unique requirements and support interaction is engineered around that single audience’s expectations Some entrepreneurs chase trending products. Others choose a niche market and drill down to find products that fit it perfectly Here are 11 markets with enough niche opportunities to support plenty of new businesses: Sustainability remains a significant concern for consumers. Every year, surveys show that most people are willing to change their shopping habits to reduce their environmental impact. One study found 64% of shoppers rank sustainability among the most important factors when buying products, and 80% are willing to pay a premium price for eco-friendly goods. For some, buying sustainably means investing in high-quality products that will last longer. For others, it’s about adjusting habits to buy more second-hand and resale items Another type of sustainable product is vegan or cruelty-free variations of conventional items If there’s a product that’s regularly purchased by the mass market there may be a niche of conscious consumers ready to embrace a green alternative Take Bee’s Wrap which offers a range of food wraps made from beeswax as an alternative to plastic Its products are not only environmentally friendly but cost effective for consumers Join the ranks of the world’s best online stores with Shopify’s powerful tools and features Analysts expect the global health and wellness market to reach $4.5 trillion by 2028 This industry is all about taking care of your mind If you’ve ever purchased a gluten-free brownie or an organic moisturizer you’ve contributed to the health and wellness market The success of globally recognized apps suggests the potential for small businesses to find a niche for their own wellness-based digital products From first-time sellers to global retailers Start your business journey by finding the Shopify plan that fits your needs Only Natural Pet Consumers interested in natural pet products can visit this online store and find everything they need for their pup While dogs and cats remain the most popular pets and small mammals all come with their own needs and present potential niche opportunities 🐶 Success story: How an internet-famous corgi led to a viral dog backpack biz won over hundreds of thousands of dog-lovers on Instagram His efforts to keep her safe while traveling through Manhattan led to a massively successful backpack company powered by Shopify One company that has successfully catered to a niche within the outdoor market is BioLite which makes solar-powered lights and camping stoves that turn fire into electricity—recognizing the need for sustainable energy sources for hikers Use Shopify’s domain name generator to search for business names and check domain availability instantly Recent travel trends include experiential travel, where travelers enjoy more active, immersive trips than traditional sightseeing or resort vacations. The continued rise of remote work has also led to a new breed of travelers: digital nomads who manage their careers while globetrotting Luggage brand Nomatic caters to the digital nomad niche by prioritizing efficiency, functionality, and the ability to pack light. You can see this audience focus represented across Nomatic’s entire ecommerce website Create a store and bring your idea to life with a free trial Discover how Shopify helps you manage your business and make sales Within this category, you can target niches based on popular genres like first-person shooters, or consoles like the Nintendo Switch. Shazim Mohammad launched his online store, Glorious Gaming, with products specifically aimed at PC gamers. It became a seven-figure niche business that basically runs on autopilot Shopify’s free AI business name generator will help you come up with the perfect business brand name The tech product industry is set to see significant growth in 2025, with Gartner predicting IT spend will reach a total of $5.74 trillion—that’s a 9.3% increase from 2024. This diverse group of spenders includes hardware geeks, software developers, gadget lovers, and early adopters who are passionate about cutting-edge technology. Sol is a good example of a brand in this space It sells the first wearable e-reader for book lovers who want a distraction-free reading experience. offers a unique take on digital reading by eliminating notifications and internet connectivity delivering a more immersive reading experience 🎧 PODCAST: How Headphones.com Secured a Priceless Domain Andrew Lissimore started his headphones business after seeing the impressive profit margins in consumer electronics He negotiated a deal to secure a domain name that would help his business get discovered Over the past decade, rates of homeownership in the US have struggled to surpass two-thirds of the population and the rising cost of living has created more rentals As rental properties are vacated, they often need renovations, creating market opportunities for home décor products, tools, and accessories. As homes are turned into investment properties to generate passive income one niche that’s emerged is smart security devices August keyless entry locks let owners grant temporary access to short-term renters 🥄 Success story: Old World Kitchen’s leap from Etsy to Shopify The Polder family started their handcrafted wooden spoon brand by selling door to door before launching on Etsy they sell both through the marketplace and a standalone ecommerce store Yet remote work also brings new challenges, with workers often feeling disconnected or burned out. To solve these challenges, Miro offers an online collaborative whiteboard platform that helps remote teams brainstorm bridging the gap created by physical distance Understanding the unique challenges and needs of remote workers can lead to innovative product ideas that enhance the work-from-home experience Shopify’s drag-and-drop editor makes it easy to create stunning Choose from thousands of themes and apps to help make your website one of a kind establishing a local presence can be challenging But there are ways for ecommerce entrepreneurs to successfully serve niches local to their city or country Take the apparel company Peace Collective as an example. Founded in Toronto, it initially catered to the demand for Toronto Blue Jays merchandise during a playoff run The brand has since expanded to serve other cities by securing partnerships with franchises including the NFL Only Shopify gives you all the tools you need to manage your business The global bakery market was valued at $480 billion in 2024, and is projected to grow to $721 billion by 2032. Home-baking exploded during the pandemic and the hobby stuck Chefs' Toys is a great example of a brand in this niche. The brand has a dedicated product category for bakery and pastry products with both retail locations and an online store Rather than competing head-on with bigger brands, if you specialize in a specific market segment you’ll give your business a competitive advantage from the start But how do you find your niche market? Here’s a step-by-step guide for discovering niche markets and product opportunities Start by gaining an understanding of what other online retailers are selling in a product category or to a certain audience Begin with basic Google searches for broad product categories “cruelty-free makeup” might lead you to “vegan skin care” and “not tested on animals.” Repeated trial and error can surface underserved audiences or demand in niche markets even if a competitor is targeting your niche you can still compete by niching-down on a specific segment of that audience Enter your search queries into Google Trends where you can see if a topic has steady or growing search interest over time Creating a mind map is a great way to visually explore different paths within your niche. Since mind maps mimic how our brains think they’re an intuitive way to organize your thoughts and expand on ideas Building a mind map can help generate product ideas quickly while also encouraging you to explore different niche paths. Use a free online tool like Text2MindMap to create simple but effective visual aids When you start typing a query into Google, automated suggestions appear. These are Google’s most popular related queries, which you can use to find a niche for your product category so you may want to use a keyword research tool to gather more suggested searches This practice is called long-tail keyword discovery Long-tail keywords are often composed of a larger market (i.e. “cruelty-free makeup”) followed by the word “for,” then a smaller “sensitive skin.”) They help describe the relationship between a niche and a wider product category Entrepreneurs use long-tail keyword data to assess customer interest and analyze competition in niche product areas Google’s Keyword Planner tool is similar to the Google suggestion tool above To use this tool, you’ll need a Google Ads account Log in to your account and select Tools from the top menu Enter your main niche idea to see your initial results You can adjust your location settings on the left to make sure you’re targeting areas you want to reach and check suggestions for other recommended terms Searching through keyword results can give you a good idea of potential niches related to your original search term—and sometimes even the demand for specific popular products If you don’t have a Google Ads account, you can use the Keywords Everywhere browser extension to see search volume directly under your Google searches. Discovering an appealing niche is just the beginning The next question is whether your niche has room for a new business and potential profitability of a niche is just as important as the initial idea Because few brands specialize this narrowly you’ll face fewer direct rivals and can establish a stronger mind-share faster Here are some ways to evaluate a new niche market idea: Niches with few existing competitors might seem like golden opportunities but there might be a reason other entrepreneurs have avoided that specific market Perhaps consumer demand for the niche idea isn’t high enough or products frequently have supply chain issues Before fully committing, try a small selection of dropshipping products and run a marketing campaign targeting your intended audience or send a few products to influencers for their opinion is crucial for fine-tuning your strategy before a full-scale launch and other key areas in your niche to gain further insights Can you offer a solution to a common problem What features and metrics do consumers judge products by? Keyword research helps identify a niche market but deeper analysis can reveal more actionable insights Take a look at these and other online resources to understand more about your chosen subject: Keeping abreast of developments in your niche is crucial—your business must talk and act like an expert in the know so your business might need to adapt to emerging consumer interests New product lines can be introduced as opportunities arise This flexibility will maintain your business’s relevance and profitability over time When it’s time to market your product always return to your niche audience’s needs and commonalities What makes your audience different from the broader market How can you appeal specifically to their preferences If your business can speak in your niche’s language it will have a greater chance of gaining buyer trust and winning their confidence 📚 Read: How to Conduct a Market Evaluation Your USP is what sets your business apart—a promise that resonates with customers in a personal Think of it as your brand’s superpower: the specific way you solve a problem that nobody else can dig deep into your customers’ real challenges Your most powerful USP speaks directly to a need that feels personal and urgent to your target market It’s less about features and more about value: Are you saving customers time A strong USP follows this simple formula: Help [specific people] achieve [concrete result] through [your unique approach] With your niche identified and product ideas in hand, it’s time to turn your business idea into a reality Whether your business plan involves crafting products independently, partnering with a manufacturer, or dropshipping, these resources can guide you through each step of the process: Focus on specific customer groups with unmet needs big brands overlook. Seek out passionate communities with specific challenges and look for gaps where existing solutions feel generic or impersonal. Think about markets where people feel frustrated by one-size-fits-all solutions: sustainable pet products for eco-conscious owners, adaptive fashion for people with disabilities, specialized nutrition for unique health needs, and tech accessories designed for neurodivergent professionals. A niche market is a segment of a wider market defined by specific needs or preferences that set it apart from the broader audience. A mass market targets as many people as possible, whereas a niche market serves a specific group with distinct needs or interests—like a national grocery store chain versus a local butcher shop specializing in organic meats. Identify a broad market that interests you, then drill down to find a more focused audience segment. Use Google Trends, mind maps, or social media research to validate demand. Niche marketing focuses on reaching a narrower, specialized audience with customized messaging and product offerings. It often leads to higher customer loyalty and less direct competition. Use market research to gauge trends, demand, and competition. Google Trends, keyword research tools, and social media can provide valuable insights. Validate by measuring potential growth and profitability. Join millions of self-starters in getting business resources Unsubscribe anytime. By entering your email, you agree to receive marketing emails from Shopify. By proceeding, you agree to the Terms and Conditions and Privacy Policy. Unsubscribe anytime. By entering your email, you agree to receive marketing emails from Shopify. By proceeding, you agree to the Terms and Conditions and Privacy Policy and explore all the tools you need to start Climate change is challenging the limits of traditional insurance by creating new risks and exacerbating old ones at an unprecedented scale As weather events increase in frequency and severity many businesses find themselves underinsured or unable to secure coverage Arbol addresses these growing protection gaps with parametric insurance which quickly assesses damage based on a predetermined set of markers informed by historical data In an interview with McKinsey’s Fabian Metzeler discusses how parametric insurance creates extra value in the insurance industry Arbol’s obsessive focus on cashflow over growth why AI has been essential to streamlining Arbol’s sales process and operations and what he wishes he’d done differently while fundraising for the company Key insight #1: Parametric insurance supplements traditional insurance in situations and industries that are challenging to cover could you explain the difference between parametric insurance and the more traditional insurance products we’re familiar with Why is parametric insurance specifically gaining momentum in areas that are highly exposed to weather-related risks Siddhartha Jha is the co-founder and CEO of Arbol a decentralized marketplace for climate data and has more than 13 years of experience in commodity trading and quantitative strategies Jha holds both bachelor’s and master’s degrees in applied mathematics and economics from Harvard University Siddhartha Jha: Parametric insurance is starting to gain traction as people become more aware of it The primary difference is that parametric insurance is triggered by a data point rather than a subjective loss assessment an adjuster or specialist comes to your property after a natural disaster like a flood or hurricane This subjectivity can introduce a number of inefficiencies and sometimes even fraud into the claims process It also makes scaling coverage a challenge More parts of the world and types of insurance have reduced coverage or are experiencing an increase in delayed payments people are unsure when and how much they’ll get paid for their claim Parametric insurance tries to address those challenges by using increasingly granular data sets to help with this process especially when traditional insurance lacks sufficient coverage or has many exclusions climate insurance is an ideal parametric use case because we continue to collect more precise data for the weather and climate Fabian Metzeler: Could you give an illustrative example as to how parametric insurance can be used in an industry such as agriculture or energy Siddhartha Jha: An example in agriculture might be when a farmer is worried about deficient rainfall over a crucial growing period for their crop Parametric insurance would use a highly granular rainfall measurement that applies to their farm area If rainfall is 50 percent below a long-term average This also could apply to a processor that makes flour They might be exposed to a drought in the areas where they source the wheat from Processors aren’t growing the crop directly so it’s difficult to get insurance for their supply chain especially if it includes thousands of farms There wouldn’t be enough adjusters to cover the area could easily cover a district or a number of districts It starts to add value in situations where sending an adjuster would be too difficult a utility that is worried about a summer heat wave but they may be limited to a certain amount for that period because their plants only produce so much If they have to deliver more power unexpectedly they can’t cut power because that will be costly in other ways they have to procure more power in the live spot market or possibly a hundred times more than average utilities often buy temperature contracts to protect themselves That way if there’s an unexpected heat wave they get a payout that helps cover some of the cost of procuring spot power Key insight #2: Instead of challenging incumbents leverage your perspective as an outsider to identify and solve for gaps in the industry—then prove you can execute Fabian Metzeler: How did you come up with the idea to found a new company in this highly competitive space crowded with big competent players with huge financial reserves Founded in 2018 and headquartered in New York Arbol is an innovative insurtech company operating in more than 15 countries and specializing in data-driven parametric insurance solutions for climate risks Arbol closed a $60 million Series B funding round with Giant Ventures and Opera Tech Ventures Siddhartha Jha: There was a confluence of factors One was that I was an outsider who didn’t overthink the problem I was from the commodities world where weather and climate are big problems So we started with a blank slate when it came to thinking about climate impacts in an insurance context The makeup of our team was crucial to tackling this problem We assembled a team that had expertise in new technologies Our founding team’s position wasn’t to beat the incumbents but rather to figure out where there were gaps in coverage We asked what sort of products a farmer or a utility would need besides traditional insurance Then we brought new players into the market who could service those needs because those gaps weren’t being filled by incumbents due to the risk can put you in a better position to navigate funding fluctuations in the market Fabian Metzeler: The heyday for start-ups is How did you and Arbol navigate the sudden paradigm shift from “growth at all costs” to “better become profitable ASAP” In which areas did you need to pivot to adopt to this change of direction Leap by McKinsey works with established organizations to imagine and scale new businesses—and develop the capabilities needed to do it again and again We bring together a global network of experts to build dynamic innovative businesses that can reinvigorate entire organizations Learn more about Leap by McKinsey Siddhartha Jha: My co-founder had been in the markets since the late 1980s while I spent the first five years or so of my career in interest rates and macroeconomics We approached fundraising with a different viewpoint from someone who has a purely tech start-up background We never followed the growth-at-all-costs model and were therefore out of sync with the fundraising market during those years I remember that our seed round deck talked about when we would achieve positive cash flow because we knew that the interest rate cycle had to change at some point and funding would dry up Our focus was confusing to prospective investors We obsessively focused on how every dollar was spent and how much revenue it could generate we were in a much better spot compared with many other firms that had overly emphasized the growth aspect Fabian Metzeler: How did you navigate the transition from “scrappy” to “professional” as a fast-growth start-up how did you time and sequence the maturation of organization while retaining the culture and agility especially in an industry with a limited appetite for experimentation Siddhartha Jha: One of the things we’ve focused on is hiring a mix of entrepreneurial and deeply experienced people who can take on different aspects of our roles they tend to become less entrepreneurial and more entrenched in their roles we try to have someone start off in a deputy role to learn the ropes Then they can start taking on more responsibilities We’re still fostering this independence in areas like sales efforts where we want to expand past founder-led strategic partnerships into having people who build partnerships on their own It’s difficult to train people in business development because it includes educating customers on the product and checking all the legal and regulatory boxes But that’s where building a cohesive team who can work with each other and think outside the box is so crucial Key insight #4: Deliberately build a technology stack based on how it generates revenue and reduces cost to streamline your operations Fabian Metzeler: How did AI help you work with the large and complex sets of data parametric insurance is built upon Siddhartha Jha: We’ve used AI since the start of building the company My own background is in graduate-level statistics and we have a deep bench of expertise in AI and risk management to be an AI engine that has been learning since day one This was not a “nice to have” element of our process; it was a necessity given that climate dynamics are incredibly complex and produce vast amounts of data we have tens if not hundreds of millions of data points that have to be processed and understood a climate event happening in East Asia can affect another one happening in West Africa It is nearly impossible for a human being to keep track of that How do you use technology to ensure you can grow in the leanest way possible And how has the strong momentum of generative AI and AI created new opportunities for you to streamline your organization Because they had an incredible array of tools at their fingertips to deploy a structure Our next area of focus has been the back-end systems and reducing human touches on the operations process That part doesn’t need AI but rather automation We’ve used automation to streamline aspects of the sales process from our early days like shifting from blockchain white paper to smart contracts We’ve built our technology stack incrementally with an eye on demonstrating its use in generating revenue or reducing costs With a “build it and they will come” approach you’ll end up with an overly engineered platform that distracts from the reason you’re in the business—to sell a product Key insight #5: Talk to everyone in your company—from top brass to rank and file—to get an on-the-ground perspective of operations and ensure that priorities are clearly understood Fabian Metzeler: How would you describe your personal management and leadership style more recent leadership team to replicate essential aspects of your leadership that are important for Arbol’s success Siddhartha Jha: The overall ethos I’ve tried to impart has been that we have the leadership team that is responsible for management we have pods that are independently responsible for their deliverables That could include a legal pod or an accounting pod but also a sales pod where you have someone who faces the client and someone who faces the risk capacity My role is to ensure that the heads of each pod understand what the priorities are and how they can cooperate with each other if the sales pod needs regulatory approval Essentially each pod is responsible for managing what equates to their own mini companies Something that’s been really helpful in navigating this vision is making a consistent effort to talk to all our employees This gives us an inside perspective on the inner workings of the company that you don’t always get from the top brass then they can’t achieve their deliverables or understand their impact Having one-on-one discussions with people from all over the firm helps make them aware of the overarching goal We have always made decisions in a group setting We recognize that everyone here is not just smart but also brings diverse and deep experience to the table A big part of my work is to distill those experiences and understand what information or expertise I’m missing from my perspective Key insight #6: Approach potential investors early to assess their expectations for investment and lay the groundwork for a successful partnership what are some pieces of advice on what to do differently that you would give to your younger self Siddhartha Jha: There are so many pieces [of advice] that it’s hard to pick One is that I would put more time and effort into hiring I’ve learned that getting the right people into the right roles doesn’t necessarily mean hiring externally Getting that right has been an important job for management to learn over time Another thing I would do differently is to be a lot of more organized about my fundraising I could have done a better job getting to know prospective investors before thinking about fundraising But even when fundraising doesn’t seem imminent it’s important to get to know investors early so that they are comfortable with you and you can assess their expectations for their investments You can also gauge what they might be like as a partner Investor partners have the potential to give you valuable advice on specific problems you might have in your business or your overall points of vulnerability The earlier you get to know your investors they earlier they can see that you can execute which lays a strong foundation for your partnership later on Siddhartha Jha is co-founder and CEO of Arbol Fabian Metzeler is a partner in McKinsey’s Düsseldorf office Comments and opinions expressed by interviewees are their own and do not represent or reflect the opinions or positions of McKinsey & Company or have its endorsement Details: cache-fra-eddf8230059-FRA 1746505758 3557935770 Metrics details Intratumour heterogeneity represents the hierarchical integration of genetic phenotypic and microenvironmental heterogeneity Although single-cell sequencing has clarified genetic and phenotypic variability microenvironmental factors remains elusive a tumour-targeted probe tracking extracellular H2O2 which allows the visualization and characterization of tumour cells exposed to oxidative stress T-AP1 identified actively budding intratumour regions as H2O2-rich microenvironments (H2O2 hotspots) which were primarily established by neutrophils tumour cells exposed to H2O2 underwent partial epithelial–mesenchymal transition through p38–MYC axis activation and migrated away from H2O2 hotspots This escape mechanism was absent in normal epithelial cells but prevalent in most cancers except NRF2-hyperactivated tumours which exhibited abrogated p38 responses and enhanced antioxidant programmes thus revealing an intrinsic stress defence programme in cancers T-AP1 enabled the identification of H2O2 hotspots that provide a niche for cancer cell dissemination offering insights into metastasis initiation Prices may be subject to local taxes which are calculated during checkout Intratumor heterogeneity: the rosetta stone of therapy resistance Unravelling biology and shifting paradigms in cancer with single-cell sequencing Non-genetic mechanisms of therapeutic resistance in cancer Therapy resistance: opportunities created by adaptive responses to targeted therapies in cancer Modulation of oxidative stress as an anticancer strategy Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment Principles of cancer therapy: oncogene and non-oncogene addiction Cancer cells co-opt the neuronal redox-sensing channel TRPA1 to promote oxidative-stress tolerance 3D culture models with CRISPR screens reveal hyperactive NRF2 as a prerequisite for spheroid formation via regulation of proliferation and ferroptosis A genetically encoded sensor for H2O2 with expanded dynamic range Antibody drug conjugate: the “biological missile” for targeted cancer therapy The oncology market for antibody–drug conjugates Trastuzumab emtansine for HER2-positive advanced breast cancer Trastuzumab deruxtecan in previously treated HER2-positive gastric cancer Molecular imaging of hydrogen peroxide produced for cell signaling The acidic tumor microenvironment as a driver of cancer Synergistic interactions between tamoxifen and trastuzumab (Herceptin) Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941 New insights into the mechanisms of epithelial–mesenchymal transition and implications for cancer Partial EMT in head and neck cancer biology: a spectrum instead of a switch Interplay between tumor microenvironment and partial EMT as the driver of tumor progression Toward clinical application of the Keap1-Nrf2 pathway Context specificity of the EMT transcriptional response and mitigate p53 function: a mechanism for oncogene-induced genetic instability The regulation and function of C-Fos and other immediate early genes in the nervous-system MAPK-regulated transcription: a continuously variable gene switch Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells Diversity and versatility of p38 kinase signalling in health and disease MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop Single-cell transcriptomic analysis of primary and metastatic tumor ecosystems in head and neck cancer Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice Soluble CD40 ligand accumulates in stored blood components and is a potential cofactor in the development of transfusion-related acute lung injury Neutrophil-activating therapy for the treatment of cancer P47phox-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene Neutrophil-derived reactive oxygen species promote tumor colonization Identification of a region in p47phox/NCF1 crucial for phagocytic NADPH oxidase (NOX2) activation Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis Oncogenic signaling pathways in The Cancer Genome Atlas Expression profiling of budding cells in colorectal cancer reveals an EMT-like phenotype and molecular subtype switching NRF2 is an upstream regulator of MYC-mediated osteoclastogenesis and pathological bone erosion Dynamic ROS control by TIGAR regulates the initiation and progression of pancreatic cancer Oxidative stress inhibits distant metastasis by human melanoma cells Nrf2 activation promotes lung cancer metastasis by inhibiting the degradation of Bach1 BACH1 stabilization by antioxidants stimulates lung cancer metastasis Neuroprotection of host cells by human central nervous system stem cells in a mouse model of infantile neuronal ceroid lipofuscinosis Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc Mechanism of early dissemination and metastasis in Her2+ mammary cancer The MYC oncogene – the grand orchestrator of cancer growth and immune evasion MYC suppresses cancer metastasis by direct transcriptional silencing of αv and β3 integrin subunits Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells Genomic characterization of human brain metastases identifies drivers of metastatic lung adenocarcinoma Circulating tumor cells exhibit metastatic tropism and reveal brain metastasis drivers A MYC and RAS co-activation signature in localized prostate cancer drives bone metastasis and castration resistance MYC levels regulate metastatic heterogeneity in pancreatic adenocarcinoma Establish a novel tumor budding-related signature to predict prognosis and guide clinical therapy in colorectal cancer Neutrophils escort circulating tumour cells to enable cell cycle progression Neutrophil dynamics in the tumor microenvironment Chronic stress increases metastasis via neutrophil-mediated changes to the microenvironment A framework for advancing our understanding of cancer-associated fibroblasts Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer Highly activatable and rapidly releasable caged fluorescein derivatives An enzymatically activated fluorescence probe for targeted tumor imaging The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth Determination of the physiological range of oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy Recommendations for reporting tumour budding in colorectal cancer based on the International Tumour Budding Consensus Conference (ITBCC) 2016 EMT subtype influences epithelial plasticity and mode of cell migration Improved vectors and genome-wide libraries for CRISPR screening Genome-scale CRISPR-Cas9 knockout screening in human cells In vivo imaging of hydrogen peroxide production in a murine tumour model with a chemoselective bioluminescent reporter An activatable cancer-targeted hydrogen peroxide probe for photoacoustic and fluorescence imaging Neighboring macrophage-induced alteration in the phenotype of colorectal cancer cells in the tumour budding area Download references Nishimura for supporting the characterization of the synthetic compounds by NMR and mass spectrometry analysis and Platforms for Advanced Technologies and Research Resources ‘Advanced Bioimaging Support’ for providing MMTV-neu/HER2 mice Irradiation exposure was conducted through the CORE Program of the Radiation Biology Center at Kyoto University This work was supported by Grant-in-Aid for Transformative Research Areas B-20H05788 (N.T.) and Scientific Research on Innovative Areas 26111004 (Y.M.) Japan Agency for Medical Research and Development under grant no the Takeda Science Foundation (N.T.) and the Cancer Foundation under grant no These authors jointly supervised this work: Shigeki Kiyonaka Department of Synthetic Chemistry and Biological Chemistry Research Institute for Quantum and Chemical Innovation Department of Clinical Bio-resource Research and Development Department of Medical Biochemistry and Biophysics Department of Breast and Endocrine Surgery performed all experiments except for the synthesis and the characterization of PG1-NHS which were performed with assistance from K.I. provided resources and participated in discussions supervised the design and measurement of T-AP1 supervised cancer biology parts and the whole project generated the first draft of the manuscript and all authors contributed to the revisions The remaining authors declare no competing interests Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations An unpaired two-tailed Mann–Whitney U-test was used to determine statistical significance Solid and dashed lines indicate tumour buds and tumour–stroma interfaces tumours (3 weeks post-inoculation) were isolated 12 h post-T-AP1 administration and data were obtained from three independent experiments Source numerical data are available in source data Source data Representative pan-cytokeratin and T-AP1 or rH-AP1 signals in the indicated tumours (two independent experiments) Representative time course of PG1/AF647 ratio changes in P-AP1 following treatment with the indicated H2O2 concentrations in HEPES buffer solution Data were normalized to pre-H2O2 values (three independent experiments) Quantification of PG1/AF647 ratio changes in P-AP1 after exposure to ROS (10 μM) for 60 min in PBS with or without 1 μg/ml catalase Percentage increases were calculated based on the values measured before ROS application An unpaired two-tailed t-test was used to determine statistical significance compared with the corresponding catalase-untreated samples Representative images of PG1/AF647 ratios in the indicated P-AP1–labelled EGFR-enriched cancer cell lines before and after 100 μM H2O2 treatment for 3 h Cells were treated with P-AP1 for 1 h followed by H2O2 in the absence of P-AP1 Values relative to corresponding H2O2-untreated cells are shown (n = 13–15) An unpaired two-tailed t-test was used to determine statistical significance relative to the corresponding H2O2-untreated cells Representative confocal images in the indicated intratumour regions in P-AP1–labelled H1838 xenograft tumours (two independent experiments) Representative confocal images in the indicated intratumour regions in E-AP1–labelled CMT-93 xenograft tumours and schematic of E-AP1–based H2O2 monitoring in vivo Quantification of PG1/AF647 ratios in the indicated regions in g (n = 29 for actively budding region and n = 30 for non-budding region) An unpaired two-tailed t-test was used to determine statistical significance Representative images of T-AP1 signals in N87 xenograft tumours harvested 1–4 weeks post-inoculation Quantification of PG1/AF647 ratios at randomly selected intratumour regions in i (n = 22–30) One-way ANOVA with Tukey’s HSD test was used to determine statistical significance Tumour bud quantification at randomly selected intratumour regions in i (n = 10 per group) A Kruskal-Wallis test with Dunn’s multiple comparison test was used to determine statistical significance Source data Data obtained from three biologically independent N87 xenograft tumours are shown as means ± SEM Statistical significance was determined using a one-sample t-test against 0 mRNA levels of NQO1 and GCLM in N87 cells treated with the indicated H2O2 concentrations for 24 h Data were obtained from three independent experiments normalized to cells not treated with H2O2 and are shown as means ± SEM Representative FCM plots of T-AP1–labelled N87 xenograft tumour cells stained with the indicated antibodies (three independent experiments) Thresholds for expression levels were defined based on IgG-AF546 controls (red and green gates) Representative FCM plots of the PG1- and AF647-derived fluorescence in T-AP1–labelled PDX000074 tumour cells stained with MYC VIM or E-CAD antibodies (two independent experiments) Quantification of PG1/AF647 ratios in the indicated AF647+ tumour cells from the experiments described in g Representative FCM plots of the PG1- and AF647-derived fluorescence in T-AP1–labelled HCC1954 tumour cells stained with MYC Quantification of PG1/AF647 ratios in the indicated AF647+ tumour cells from the experiments described in i Representative confocal images of T-AP1–labelled HCC1954 xenograft tumours stained with anti-VIM antibody or anti-E-CAD antibody (two independent experiments) Solid and dashed lines mark tumour buds and tumour–stroma interfaces Data are shown as means ± SEM from one representative experiment of two independent experiments performed in triplicate Source data Immunoblot of MYC in N87 cells treated with 10 μM H2O2 VIM and E-CAD in HCC1569 cells treated with 10 μM H2O2 Wound healing assay in H2O2-treated (10 μM Number of N87 and HCC1569 cells relative to day 0 with or without 10 µM H2O2 treatment for the indicated periods Data are shown as means ± SD from one representative experiment of two independent experiments performed in triplicate Transwell migration and invasion of N87 cells pretreated with 10 μM H2O2 for 6 h or untreated Mean ( ± SEM) number of migrating or invading cells normalized to untreated cells is also shown Matrigel™ was applied to the upper membrane in the invasion assay 36 h) or untreated N87 cells transduced with sgControl The average percentage of the wound closure is also shown VIM and E-CAD in N87 cells transfected with a MYC-expressing or empty vector and treated with water (control) or 1 mM NAC for 24 h PG1/AF647 ratios in N87 cells transfected with a MYC-expressing or empty vector and treated with T-AP1 for 1 h followed by T-AP1–free complete medium for 12 h (n = 21 and 24 for the vector and MYC from the sum of two independent experiments) Schematic for MYC knockdown induction in tumours using doxycycline MYC mRNA levels in N87 cells transduced with inducible shRNAs (shMYC-#1 shMYC-#2) or control (shControl) after 72 h doxycycline treatment (2 μg ml−1) Data were normalized to shControl-transduced cells and are shown as means ± SD from one representative experiment of two independent experiments performed in triplicate MYC mRNA levels in shControl- or shMYC-transduced N87 xenograft tumours (3 weeks post-inoculation) Mice were treated with doxycycline (2 mg ml−1 in drinking water) for 1 week as described in i Data were normalized to shControl-transduced tumour and are shown as means ± SD from one representative experiment of two independent experiments performed in triplicate Volumes of the shControl- or shMYC-transduced N87 xenograft tumours (3 weeks post-inoculation) prepared as described in i Representative confocal images of pan-cytokeratin signals in shControl- or shMYC-transduced N87 xenograft tumours stained with anti-pan-cytokeratin antibody and prepared as described in i Quantification of tumour buds at randomly selected intratumour regions from the experiment described in m (n = 15 per group from the sum of three independent experiments) Mean ( ± SEM) fluorescence intensity (MFI) of VIM-AF647 in RFP+ tumour cells isolated from shControl- or shMYC-transduced N87 xenograft tumours Cells were stained with an antibody raised in rabbit against VIM followed by an AF647-conjugated secondary antibody against rabbit IgG and subjected to FCM analysis data were obtained from three independent experiments Source numerical data and unprocessed blots are available in source data Source data Representative confocal images of T-AP1–labelled N87 xenograft tumours treated with HypoxyprobeTM (pimonidazole) to label hypoxic regions (anti-pimonidazole staining) from two independent experiments Pimonidazole was injected 1 h before tumour resection Representative confocal images of T-AP1–labelled N87 xenograft tumours stained with anti-HIF-1α antibody (two independent experiments) GSEA of RNA-seq data comparing PG1highAF647+ and PG1lowAF647+ cells for TGF-β signalling genes Representative confocal images of T-AP1–labelled N87 xenograft tumours stained with anti-TGF-β antibody (two independent experiments) Representative confocal images of T-AP1–labelled N87 xenograft tumours (2 or 4 weeks post-inoculation) stained with anti-F4/80 to identify macrophages (three independent experiments) Quantification of F4/80+ area at randomly selected intratumour regions from the experiments described in e (n = 15 per group from the sum of three independent experiments) Correlation between macrophage accumulation and PG1/AF647 ratio at randomly selected intratumour regions in the T-AP1–labelled N87 xenograft tumours (4 weeks post-inoculation) from the experiments described in e (n = 30 from the sum of three independent experiments) Representative confocal images of actively budding or non-budding intratumour regions in the indicated xenograft tumours labelled with T-AP1 P-AP1 or rH-AP1 and stained with anti-Ly6G (two or three independent experiments) Correlations between the PG1/AF647 ratio and Ly6G + neutrophil accumulation at randomly selected intratumour regions in H1568 (n = 39) and PDX000074 xenograft tumours (n = 33) from the experiments described in h Correlations between the PG1/AF647 ratio and Gr-1+ neutrophils CD4+ or CD8+ T cells or NK1.1+ natural killer cells at randomly selected intratumour regions in rH-AP1–labelled tumours from MMTV-HER2/neu mice (n = 22 respectively from the sum of two independent experiments) Source data This analysis was performed across 225 patient samples Source data Frequency of budding cells per total tumour cells calculated from images of pan-cytokeratin immunohistochemistry in tumour sections from patients with colorectal (n = 252) lung (n = 191) or stomach cancer (n = 210) The number of total tumour cells in each tissue microarray core was counted using an automatic particle counting program in ImageJ based on the double positivity in nucleus/pan-cytokeratin staining The number of budding tumour cells were counted manually Representative images of NQO1 and pan-cytokeratin immunohistochemistry in serial tumour sections from patients with lung cancer (b) (n = 191) and stomach cancer (c) (n = 210) Tumours with a budding cell frequency equal to or above the median value (that is 1.061 for lung cancer or 3.557 for stomach cancer) were defined as high-grade budding while those below the median were defined as low-grade budding Correlation between the NRF2 score and budding signature score in lung adenocarcinoma (n = 515) and squamous cell carcinoma data (n = 502) obtained from TCGA The two-sided P value was calculated using Pearson’s r Source data Quantification of PG1/AF647 ratios in non-budding intratumour regions from the experiments described in b (sgControl: n = 28 mRNA levels of the indicated mesenchymal marker genes in N87 cells transduced with sgControl or sgKEAP1 and treated with the indicated concentrations of H2O2 for 6 h Data were normalized to cells not treated with H2O2 The sum of H2O2-induced log2 fold changes in the expression of mesenchymal markers (VIM TNC and CDH11) was defined as the mesenchymal shift score Immunoblot of indicated proteins in sgControl- or sgKEAP1-transduced N87 cells treated with or without H2O2 (10 or 45 μM) for the indicated periods Changes in HyPer-2 ratio in N87 cells transduced with HyPer-2 and either sgControl or sgKEAP1 upon treatment with H2O2 (10 Statistical significant between sgControl- and sgKEAP1-transduced cells was determined by An unpaired two-tailed t-test Statistical significance was determined by an unpaired two-tailed t-test Source data mRNA levels of KEAP1 in the indicated cancer cells transduced with SMARTvector-inducible shRNA targeting KEAP1 (shKEAP1-#1 or shKEAP1-#2) or a non-targeting control (shControl) Cells were treated with 2 μg ml−1 doxycycline for 72 h Data were normalized to shControl-transduced cells mRNA levels of KEAP1 in the shControl- or shKEAP1-transduced N87 xenograft tumours Mice were treated with doxycycline as described in the Methods Data were normalized to shControl-transduced tumour Volumes of the indicated xenograft tumours (8 weeks post-inoculation) transduced with shControl Representative pan-cytokeratin signals in the indicated xenograft tumours transduced with shControl shKEAP1-#1 or shKEAP1-#2 from mice treated with doxycycline as described in the Methods Quantification of tumour buds in d (n = 15 per group) Representative pan-cytokeratin signals in the indicated xenograft tumours from mice injected with or without catalase as described in the Methods Quantification of tumour buds in f (n = 15 per group) Representative images of Ku80 signals in liver sections from mice bearing the indicated tumours transduced with shControl shKEAP1-#1 or shKEAP1-#2 and treated with doxycycline as described in the Methods NQO1 staining scores in tumour tissues from patients with colorectal (n = 238 Statistical significance was determined by Chi-squared test Data are shown as means ± SD from one representative of two independent experiments performed in triplicate and statistical significance was determined by an unpaired two-tailed t-test Statistical significance was determined by an unpaired two-tailed Mann–Whitney U-test Source data Volumes of the indicated xenograft tumours (8 weeks post-inoculation) in mice injected with NAC catalase or PBS from three independent experiments Mice were treated with NAC (1 mg in 200 μl PBS) catalase (100 μg in 100 μl PBS) or vehicle (100 μl PBS) twice daily for 2 weeks Representative confocal images of Ku80 signals in liver sections from mice bearing the indicated xenograft tumours from three independent experiments catalase (100 μg in 100 μl PBS) or vehicle (100 μl PBS) twice daily for 2 weeks until the harvest of liver (8 weeks post-inoculation) The liver sections were stained with an antibody against the human-specific nuclear marker Ku80 Diagram illustrating the dual role of ROS in metastasis Source data 1–8 and uncropped western blots for Supplementary Figures It contains RNA-seq data of PG1highAF647+ and PG1lowAF647+ cells obtained from T-AP1-labelled tumours It contains characteristics of patient samples and analysed data regarding the percentage of the CD66b+ area surrounding the tumour mass and buds NQO1 staining score and frequency of budding cells Methods information related to primer sequences Statistical Source Data for Supplementary Figs a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41556-025-01617-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research In Ask an Influencer, Business of Home explores the creator economy. This week, we spoke with Lexi Poer, the content creator behind Strolling in the Suburbs When Lexi Poer left her corporate marketing job in Atlanta and moved outside of the city to start a family She began posting on social media in 2016—a way to pass the time “When I decided to stay at home with our oldest finding this online community was my creative outlet Sharing that journey resonated with a lot of women and that was where my initial growth [online] came from.” Poer’s content came to encapsulate her entire life experience: her travels; her relationships with her mother she had watched her mom carry out a number of renovations and had inherited her love of interior design When her own family purchased a home eight years ago she knew not only that she wanted to embark on a similar project but that she wanted her mom to be part of the household as well—prompting a redesign process that’s unfolded over the last two years “In early childhood I’d always imagined it—[I was like] ‘Mom I want to live on a piece of land and have three houses: one for me one for you and one for my best friend,” says Poer my mom had a house that was probably only 20 minutes away from us but even that was enough of an inconvenience where we were like ‘Why are we doing this?’ My husband and her get along so well How can we make a better plan for the future?” In the period that followed, the experience shaped Poer’s online presence, which now centers on design and daily life for multigenerational living. Even more than her city-to-suburb transition, that unique focus has kept her audience engaged and growing, now numbering 48,500 followers on TikTok and 108,000 on Instagram and how to break through the noise to establish relationships with brands it’s way more common in other cultures to live in a multigenerational home unit,” she says ‘How can we actually do this?’— we started sharing that and there was a huge reciprocation of that concept [People were] asking all the questions that come with it but how do we make it look like you’ve made it look?’” Poer came to realize she had hit upon a trend in the U.S.: A growing number of families were embracing multigenerational households as a response to a tight housing market and the desire to spend more time with loved ones Recognizing that the topic was connecting with viewers she tapped into her marketing background to place the messaging at the forefront of her design content through hashtags and captions on posts and descriptive text in the bio of her accounts “There are so many creators who are sharing design and home What can we do that makes us stick out from that?” says Poer “Keeping that element very visible is my biggest strategy as far as making sure it’s clear what we’re trying to showcase.” She has also used online tools to help build the brand partnerships aspect of her business—in particular third-party platforms designed to help connect brands with creators based on subject matter “That is a benefit that has developed over the last several years of being members of these platforms that have the software to funnel down the creator pool to exactly what brands are looking for whether that’s a family with kids in the Atlanta area or whatever demographics the brand’s trying to reach in a campaign,” says Poer “That’s something from my corporate world that I use a lot and that I don’t think most creators are using to their advantage,” she says “I’ve been able to connect with [businesses] and get the right contacts which is usually the biggest hurdle with a lot of brands that I’ve been interested in partnering with.” Poer narrows her search for a specific contact at a large company by scanning for relevant social-media-related terms on their profile: head of brand partnerships “Usually if a brand is going to have a budget to work with creators they will have a specific person who’s managing that process,” she says “If I struggle to find someone with a title that fits typically that means they haven’t quite budgeted or put a plan in place to work with creators just yet.” She’ll send a message introducing herself and her family (“because for us how her journey started and where it is today she includes some of her social media analytics and is clear about the type of partnership and compensation she’s looking for “The more you can remove the back-and-forth the better—especially for people who are likely very busy in their roles,” says Poer “I’ve had several brand partnership managers reply I’ve never been reached out to on LinkedIn from a creator—kudos.’ I think that shows and I’m willing to show up,’ which is a big thing in our industry Not everyone is always willing to follow through with what they’re claiming they can provide so that level of professionalism and reliability actually gets to them whereas sometimes DMs get lost in the pool.” The Chickadees are chiseling away at a male-dominated craft a trio of women in matching blaze-orange parkas gathered at Quarry Road Trails to will a Greek goddess from a hunk of snow They’d started the day before with a four-by-six-foot block of packed snow emptied from a refrigerator box they chiseled from the frozen form a crude bust of Artemis etching gathers on her dress and grooves on her prodigious updo with chisels and a metal curry comb and smoothing her garment and features with sandpaper and a handleless paintbrush Sisters Serena and Phoebe Sanborn, along with Desiree Dubois, were practicing for the U.S. National Snow Sculpting Championship in Lake Geneva where they planned to render a nine-foot-tall kneeling Artemis with a bow and arrow pointed at the moon would count for three of only six women in the competition and would be the only all-female team They chose to sculpt the lunar goddess as a symbol of female strength and an homage to a planned NASA mission of the same name that would send the first woman around the moon “The physical challenge appeals to a lot of men,” Serena said “It’s important for girls to see women in spaces like this so they can feel like they can do anything.” A friend from Alaska taught Serena snow sculpting in 2013, and she began competing immediately. In 2019, she placed second in the U.S. National event with another team. “I love the challenge of collaborative problem-solving,” she said. “One brain is fine, but many brains can solve things so much easier.” Serena and Phoebe, who work for the arts organization Waterville Creates started sculpting together in 2017; a couple years later The Chickadees met weekly to sketch their Artemis sculpture and practice carving it from blocks of clay They had only one opportunity to sculpt with snow before the national competition in February Over the course of the normally frigid four-day contest participants typically spend about eight hours a day sculpting an ten-by-ten-foot block of packed snow provided by the organizers and work through the night before the final day daytime temperatures in Lake Geneva hovered in the high 30s and 40s “The first thing we noticed as the plane was landing was a landscape of brown Organizers delayed the start of the competition by a day and encouraged contestants to carve at night to avoid excessive melting “The snow behaved like the stuff that fills a snow cone,” said Dubois a sculpture of a human puppet collapsed minutes after judging concluded while the dripping tusks of a mammoth falling through ice Artemis remained intact but didn’t earn a prize having only sculpted together for a couple years and work next to teams that had been sculpting 15 The Chickadees are currently working on a sculpture of a cottage on chicken legs (home to the child-eating Baba Yaga ogress in Slavic folklore) which they plan to debut at a New Hampshire competition this winter “Everyone has their thing that is about the inner challenge proving to yourself that you can do it,” Phoebe said “Snow sculpting gives us such a feeling of accomplishment Metrics details tumour cells integrate with the surrounding normal cells to form an abnormal structure that is tightly integrated with the host organism via blood and lymphatic vessels and even neural associations emerging cancers send a plethora of mediators that efficiently perturb the entire organism and induce changes in distant tissues These perturbations serendipitously favour early metastatic establishment by promoting a more favourable tissue environment (niche) that supports the persistence of disseminated tumour cells within a foreign tissue Because the establishment of early metastatic niches represents a key limiting step for metastasis the creation of a more suitable pre-conditioned tissue strongly enhances metastatic success we provide an updated view of the mechanisms and mediators of primary tumours described so far that induce a pro-metastatic conditioning of distant organs which favours early metastatic niche formation We reflect on the nature of cancer-induced systemic conditioning considering that non-cancer-dependent perturbations of tissue homeostasis are also able to trigger pro-metastatic conditioning We argue that a more holistic view of the processes catalysing metastatic progression is needed to identify preventive or therapeutic opportunities The role of extracellular vesicles in cancer Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response Tumor innervation: peripheral nerves take control of the tumor microenvironment Cancer as a homeostatic challenge: the role of the hypothalamus A role for hypocretin/orexin in metabolic and sleep abnormalities in a mouse model of non-metastatic breast cancer Neutrophils in cancer: heterogeneous and multifaceted Venous thromboembolism and cancer: a comprehensive review from pathophysiology to novel treatment Cancer-associated pathways and biomarkers of venous thrombosis MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche this pioneering study demonstrates the ability of cancer cells to create a hospitable environment at distant 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niche via prostaglandin E2 Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci Tissue inhibitor of metalloproteinases (TIMP)‐1 creates a premetastatic niche in the liver through SDF‐1/CXCR4‐dependent neutrophil recruitment in mice Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity Macrophage-secreted S100A4 supports breast cancer metastasis by remodeling the extracellular matrix in the premetastatic niche Mesenchymal cancer cell–stroma crosstalk promotes niche activation Primary tumors release ITGBL1-rich extracellular vesicles to promote distal metastatic tumor growth through fibroblast-niche formation KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs Extracellular mRNA transported to the nucleus exerts translation-independent function Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts Osteosarcoma-derived extracellular vesicles induce lung fibroblast reprogramming Tumor-derived Cav-1 promotes pre-metastatic niche formation and lung metastasis in breast cancer Mutant p53s generate pro-invasive niches by influencing exosome podocalyxin levels Highly‐metastatic colorectal cancer cell released miR‐181a‐5p‐rich extracellular vesicles promote liver metastasis by activating hepatic stellate cells and remodelling the tumour microenvironment Exosome-delivered CD44v6/C1QBP complex drives pancreatic cancer liver metastasis by promoting fibrotic liver microenvironment Bone-marrow-derived cell-released extracellular vesicle miR-92a regulates hepatic pre-metastatic niche in lung cancer Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche Matrix stiffness-upregulated LOXL2 promotes fibronectin production MMP9 and CXCL12 expression and BMDCs recruitment to assist pre-metastatic niche formation The pathological significance of LOXL2 in pre-metastatic niche formation of HCC and its related molecular mechanism Breast cancer-secreted factors promote lung metastasis by signaling systemically to induce a fibrotic premetastatic niche Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment Pancreatic premalignant lesions secrete tissue inhibitor of metalloproteinases-1 which activates hepatic stellate cells via CD63 signaling to create a premetastatic niche in the liver Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis Exosome‐delivered miR‐221/222 exacerbates tumor liver metastasis by targeting SPINT1 in colorectal cancer Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts Tumor-secreted exosomal miR-141 activates tumor–stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis Metastatic niches and the modulatory contribution of mesenchymal stem cells and its exosomes Lipid-laden lung mesenchymal cells foster breast cancer metastasis via metabolic reprogramming of tumor cells and natural killer cells Lung mesenchymal cells elicit lipid storage in neutrophils that fuel breast cancer lung metastasis Lung mesenchymal stromal cells influenced by Th2 cytokines mobilize neutrophils and facilitate metastasis by producing complement C3 Extracellular vesicles promotes liver metastasis of lung cancer by ALAHM increasing hepatocellular secretion of HGF Hepatocytes direct the formation of a pro-metastatic niche in the liver Tumor exosomal RNAs promote lung pre-metastatic niche formation by activating alveolar epithelial TLR3 to recruit neutrophils This key study identifies epithelial cells as active regulators of immune responses in the context of tumour-induced PMN formation Breast tumor-derived exosomal microRNA-200b-3p promotes specific organ metastasis through regulating CCL2 expression in lung epithelial cells Tumor-polarized GPX3 + AT2 lung epithelial cells promote premetastatic niche formation A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling This key study demonstrates how distal tumours shape the metabolic microenvironment of the PMN through increased local production of palmitate to support subsequent metastatic growth Fatty acid metabolism of immune cells: a new target of tumour immunotherapy Targeting metastasis-initiating cells through the fatty acid receptor CD36 Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis Hepatoma cell‐secreted exosomal microRNA‐103 increases vascular permeability and promotes metastasis by targeting junction proteins Cancer-derived exosomal miR-25-3p promotes pre-metastatic niche formation by inducing vascular permeability and angiogenesis Extracellular vesicle-mediated purinergic signaling contributes to host microenvironment plasticity and metastasis in triple negative breast cancer Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding Endothelial Notch1 activity facilitates metastasis Nidogen 1‐enriched extracellular vesicles facilitate extrahepatic metastasis of liver cancer by activating pulmonary fibroblasts to secrete tumor necrosis factor receptor 1 Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs Bone vascular niche E-selectin induces mesenchymal–epithelial transition and Wnt activation in cancer cells to promote bone metastasis Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis TGF-β1-mediated exosomal lnc-MMP2-2 increases blood–brain barrier permeability via the miRNA-1207-5p/EPB41L5 axis to promote non-small-cell lung cancer brain metastasis A blazing landscape: neuroinflammation shapes brain metastasis Lung cancer cells-controlled Dkk-1 production in brain metastatic cascade drive microglia to acquire a pro-tumorigenic phenotype Lymphangiogenesis and lymphatic vessel remodelling in cancer Lymph node colonization induces tumor-immune tolerance to promote distant metastasis Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization Melanoma‐derived extracellular vesicles mediate lymphatic remodelling and impair tumour immunity in draining lymph nodes Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis Exosomal ANGPTL1 attenuates colorectal cancer liver metastasis by regulating Kupffer cell secretion pattern and impeding MMP9 induced vascular leakiness Hypoxia‐inducible exosomes facilitate liver‐tropic premetastatic niche in colorectal cancer miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche Monocyte-derived Kupffer cells dominate in the Kupffer cell pool during liver injury Redefining macrophage and neutrophil biology in the metastatic cascade Biology of lung macrophages in health and disease High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health CNS-native myeloid cells drive immune suppression in the brain metastatic niche through Cxcl10 Compensatory CSF2-driven macrophage activation promotes adaptive resistance to CSF1R inhibition in breast-to-brain metastasis Patrolling monocytes control tumor metastasis to the lung Patrolling monocytes inhibit osteosarcoma metastasis to the lung Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche Citrullinated fibrinogen–SAAs complex causes vascular metastagenesis Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice Tumor-derived exosomal miR-934 induces macrophage M2 polarization to promote liver metastasis of colorectal cancer Gastric cancer-derived exosomal miR-519a-3p promotes liver metastasis by inducing intrahepatic M2-like macrophage-mediated angiogenesis Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming Innate lymphoid cells and innate-like T cells in cancer—at the crossroads of innate and adaptive immunity Type 2 innate lymphoid cells impede IL-33-mediated tumor suppression ILC2-driven innate immune checkpoint mechanism antagonizes NK cell antimetastatic function in the lung The changing role of natural killer cells in cancer metastasis Impairment of NKG2D-mediated tumor immunity by TGF-β TGFβ drives NK cell metabolic dysfunction in human metastatic breast cancer Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells Platelet–cancer interplay: molecular mechanisms and new therapeutic avenues Tumor-derived exosomes induce the formation of neutrophil extracellular traps: implications for the establishment of cancer-associated thrombosis Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2 Platelet TSP-1 controls prostate cancer-induced osteoclast differentiation and bone marrow-derived cell mobilization through TGFβ-1 Platelets govern pre-metastatic tumor communication to bone Neutrophils support lung colonization of metastasis-initiating breast cancer cells Neutrophil phenotypes and functions in cancer: a consensus statement Neutrophil plasticity in the tumor microenvironment Radiation exposure elicits a neutrophil-driven response in healthy lung tissue that enhances metastatic colonization Co-option of neutrophil fates by tissue environments Defining the emergence of myeloid-derived suppressor cells in breast cancer using single-cell transcriptomics Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice Identification of an early unipotent neutrophil progenitor with pro-tumoral activity in mouse and human bone marrow Developmental analysis of bone marrow neutrophils reveals populations specialized in expansion Coexpression of CD71 and CD117 identifies an early unipotent neutrophil progenitor population in human bone marrow Extramedullary hematopoiesis secondary to malignant solid tumors: a case report and literature review Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis Breast cancer remotely imposes a myeloid bias on haematopoietic stem cells by reprogramming the bone marrow niche An IL-4 signalling axis in bone marrow drives pro-tumorigenic myelopoiesis This key study provides important mechanistic insight into myelopoiesis in cancer including the identification of an IL-4-dependent axis within the bone marrow that directs GMP fate towards the production of monocytes and macrophages imprinted with immunosuppressive phenotypes thus offering novel routes to potential therapies Osteoblasts remotely supply lung tumors with cancer-promoting SiglecF high neutrophils Loss of p53 triggers Wnt-dependent systemic inflammation to drive breast cancer metastasis This important study demonstrates how tumour-intrinsic heterogeneity has important implications for the generation of pro-metastatic systemic inflammatory responses Osteoprogenitor–GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal Monocyte-derived macrophages promote breast cancer bone metastasis outgrowth Bone marrow/bone pre-metastatic niche for breast cancer cells colonization: the role of mesenchymal stromal cells RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis Mammary tumour cells remodel the bone marrow vascular microenvironment to support metastasis Association of bone metastasis with early-stage breast cancer in women with and without precancer osteoporosis according to osteoporosis therapy status Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation Unique pattern of neutrophil migration and function during tumor progression Fatty acid transport protein 2 reprograms neutrophils in cancer Tumour-elicited neutrophils engage mitochondrial metabolism to circumvent nutrient limitations and maintain immune suppression Dual roles of neutrophils in metastatic colonization are governed by the host NK cell status DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25 This study offers key mechanistic insights into how cancer cells detect and actively respond to NETs within the PMN Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum FGF19‐induced inflammatory CAF promoted neutrophil extracellular trap formation in the liver metastasis of colorectal cancer HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis ETV4 mediated tumor‐associated neutrophil infiltration facilitates lymphangiogenesis and lymphatic metastasis of bladder cancer Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice IL-1β inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization Neutrophil extracellular traps drive epithelial–mesenchymal transition of human colon cancer Neutrophils promote tumor invasion via FAM3C-mediated epithelial-to-mesenchymal transition in gastric cancer FAM3C in circulating tumor-derived extracellular vesicles promotes non-small cell lung cancer growth in secondary sites Evaluation and management of body composition changes in cancer patients Cachexia: a systemic consequence of progressive Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination Extracellular vesicle-mediated interorgan communication in metabolic diseases Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis Cancer-cell-secreted extracellular vesicles suppress insulin secretion through miR-122 to impair systemic glucose homeostasis and contribute to tumour growth Tumour extracellular vesicles and particles induce liver metabolic dysfunction This landmark study uncovers how tumour-derived EVs disrupt broader host physiology by inducing systemic metabolic alterations through EV-mediated perturbations of hepatocyte function Cancer-cell-secreted miR-204-5p induces leptin signalling pathway in white adipose tissue to promote cancer-associated cachexia An immune-sympathetic neuron communication axis guides adipose tissue browning in cancer-associated cachexia Gut microbiota in human metabolic health and disease The human intestinal microbiome in health and disease Cancer induces a stress ileopathy depending on B-adrenergic receptors and promoting dysbiosis that contributes to carcinogenesis Neutrophil ageing is regulated by the microbiome An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors Microbiota and muscle highway—two-way traffic Targeting the gut and tumor microbiota in cancer Molecular crosstalk between circadian clock and cancer and therapeutic implications Lung adenocarcinoma distally rewires hepatic circadian homeostasis This study identifies the circadian rhythm as a biological process affected by cancer cells (specifically lung cancer cells) within the primary tumour and the consequent disruption of liver metabolism The metastatic spread of breast cancer accelerates during sleep This landmark study reveals the impact of chronic stress an inevitable aspect of a cancer diagnosis highlighting how stress-induced glucocorticoids induce pro-metastatic neutrophil phenotypes to create a pro-metastatic tissue microenvironment Reactivation of dormant tumor cells by modified lipids derived from stress-activated neutrophils Neutrophil oxidative stress mediates obesity-associated vascular dysfunction and metastatic transmigration Nicotine promotes breast cancer metastasis by stimulating N2 neutrophils and generating pre-metastatic niche in lung Interorgan communication with the liver: novel mechanisms and therapeutic targets Hepatocytes derived increased SAA1 promotes intrahepatic platelet aggregation and aggravates liver inflammation in NAFLD Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice Chronic psychological stress promotes breast cancer pre-metastatic niche formation by mobilizing splenic MDSCs via TAM/CXCL1 signaling Stromal changes in the aged lung induce an emergence from melanoma dormancy Age-associated microenvironmental changes highlight the role of PDGF-C in ER+ breast cancer metastatic relapse This important study uncovers a key role for ageing-associated changes to the tissue microenvironment in regulating the metastatic dormancy of breast cancer Cellular senescence in ageing: from mechanisms to therapeutic opportunities Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage Age-associated myeloid biased hematopoiesis depends on relative decrease of short-term hematopoietic stem cell Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis Modulation of intestinal microbiota by glycyrrhizic acid prevents high-fat diet-enhanced pre-metastatic niche formation and metastasis Capsaicin shapes gut microbiota and pre-metastatic niche to facilitate cancer metastasis to liver The bone marrow protects and optimizes immunological memory during dietary restriction Control of immunity via nutritional interventions An exercise-induced metabolic shield in distant organs blocks cancer progression and metastatic dissemination Exercise training improves tumor control by increasing CD8+ T-cell infiltration via CXCR3 signaling and sensitizes breast cancer to immune checkpoint blockade Potential roles of muscle-derived extracellular vesicles in remodeling cellular microenvironment: proposed implications of the exercise-induced myokine Brown-fat-mediated tumour suppression by cold-altered global metabolism Hallmarks of sex bias in immuno-oncology: mechanisms and therapeutic implications Tumor-suppressor genes that escape from X-inactivation contribute to cancer sex bias Racial and ethnic disparities in a state‐wide registry of patients with pancreatic cancer and an exploratory investigation of cancer cachexia as a contributor to observed inequities Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models Neutrophil extracellular traps formed during chemotherapy confer treatment resistance via TGF-β activation Chemotherapy-induced complement signaling modulates immunosuppression and metastatic relapse in breast cancer Reactive myelopoiesis and FX-expressing macrophages triggered by chemotherapy promote cancer lung metastasis Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer TRPM2 mediates neutrophil killing of disseminated tumor cells Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model Tumour exosome integrins determine organotropic metastasis This landmark study uncovers how organ specificity of metastases can be generated by tumour-intrinsic mechanisms involving release of exosomes with organ-homing properties determined by the matched integrin expression and ECM make-up of target tissues Regulation of cargo selection in exosome biogenesis and its biomedical applications in cancer Estradiol induces BDNF/TrkB signaling in triple-negative breast cancer to promote brain metastases Cerebral metastases in metastatic breast cancer: disease-specific risk factors and survival Epigenetic therapy inhibits metastases by disrupting premetastatic niches This important study demonstrates how epigenetic therapies can be leveraged to reprogramme pro-metastatic cancer-primed myeloid cells and prevent PMN formation Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis This groundbreaking study demonstrates how the inflammatory characteristics of the PMN can be harnessed to develop an engineered myeloid-cell-based therapy that reprogrammes the tissue microenvironment through targeted cytokine production and effectively reverses PMN formation It offers proof-of-concept for using cell-based therapies to deliver therapeutic cargo into the PMN to prevent metastasis Remodeling of metastatic vasculature reduces lung colonization and sensitizes overt metastases to immunotherapy An interferon-driven oxysterol-based defense against tumor-derived extracellular vesicles Beta blockers and breast cancer mortality: a population- based study Beta blockers and improved progression-free survival in patients with advanced HER2 negative breast cancer: a retrospective analysis of the ROSE/TRIO-012 study Tumor-on-chip modeling of organ-specific cancer and metastasis Three-dimensional human liver-chip emulating premetastatic niche formation by breast cancer-derived extracellular vesicles A multi‐site metastasis‐on‐a‐chip microphysiological system for assessing metastatic preference of cancer cells Engineering the early bone metastatic niche through human vascularized immuno bone minitissues Multiorgan-on-a-chip: a systemic approach to model and decipher inter-organ communication A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs Biomaterial scaffolds as pre‐metastatic niche mimics systemically alter the primary tumor and tumor microenvironment Extracellular matrix mediators of metastatic cell colonization characterized using scaffold mimics of the pre-metastatic niche Sponge-like nano-system suppresses tumor recurrence and metastasis by restraining myeloid-derived suppressor cells-mediated immunosuppression and formation of pre-metastatic niche Versatile ginsenoside Rg3 liposomes inhibit tumor metastasis by capturing circulating tumor cells and destroying metastatic niches Tenascin-C expression in the lymph node pre-metastatic niche in muscle-invasive bladder cancer Circulating neutrophils from patients with early breast cancer have distinct subtype-dependent phenotypes Liquid biopsy in pre-metastatic niche: from molecular mechanism to clinical application Single particle automated Raman trapping analysis of breast cancer cell-derived extracellular vesicles as cancer biomarkers Computed tomography reveals microenvironment changes in premetastatic lung Whole-liver enhanced CT radiomics analysis to predict metachronous liver metastases after rectal cancer surgery Radiomics analysis of lung CT image for the early detection of metastases in patients with breast cancer: preliminary findings from a retrospective cohort study Tumor entrained neutrophils inhibit seeding in the premetastatic lung Download references The authors gratefully acknowledge funding support from a European Research Council grant (ERC CoG-H2020725492) and the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001112) the UK Medical Research Council (FC001112) These authors contributed equally: Nicolas Rabas The authors contributed equally to researched data for the article All authors contributed substantially to discussion of the content wrote and reviewed parts of the manuscript before submission The authors declare no competing interests for their contribution to the peer review of this work Specialized cells of the adipose tissue derived from mesenchymal stem cells that function as precursors of adipocytes Process by which osteoclasts dissolve the minerals in bone and break down the matrix Complex multiorgan syndrome causing wasting of the body Animal model of depression and stress characterized by exposure to a combination of mild and randomly administered sources of stress such as food and water deprivation The purpose of this model is to mimic the stress in daily human life Process by which the amino acid arginine is converted into citrulline by post-translational modifications catalysed by peptidyl arginine deaminases Extracellular matrix from biological origins that have been processed to remove their cellular components to obtain a scaffold of the site-specific extracellular protein composition Action carried out by specialized cells that is characterized by the release of intracellular granules Haematological process whereby granulocytes — namely neutrophils This process is enhanced under certain inflammatory contexts and termed emergency granulopoiesis High levels of blood glucose in the absence of external glucose sources (such as food) during periods of fasting Metabolic process to mobilize energy via the hydrolysis of triglycerides into glycerol and free fatty acids RNA sequences usually of about 200 nucleotides that are not translated into proteins Physiological process for the formation and growth of new lymphatic vessels from pre-existing ones Chemical and biophysical composition of the extracellular matrix The microbiota naturally living in the human body at a given location or habitat The term usually used in the context of the intestine is ‘gut microbiome’ Resident macrophages of the nervous system Biological process for the formation of all myeloid cells (such as monocytes and granulocytes) from undifferentiated bone marrow progenitor cells usually characterized by the expression of α-smooth-muscle actin and exhibiting a smooth-muscle (spindle) morphology Anatomical structure that provides integrity and regulates the blood–brain barrier Composed of cerebral vascular cells and surrounding neurons An extracellular network of decondensed DNA and proteases from neutrophils usually formed in the context of pathogens The neutrophil specific process of NET release is known as NETosis A multi-layered specialized tissue of the peritoneum Specialized cells originating from the monocyte lineage in the bone marrow whose function is to synthesize bone tissue A specialized cell originating from the monocyte lineage in the bone marrow whose function is to break down bone tissue The biological processes involved in bone degradation including osteoclast differentiation and bone resorption Neuroendocrine pancreatic cells responsible for insulin production Extracellular signalling mediated by purine nucleotides Vascular obstruction due to the formation of a blood clot The most abundant type of adipose tissue involved in energy storage endocrine communication and insulin sensitivity Metabolic adaptation to increased thermogenic demand characterized by the development of brown adipose tissue or the conversion of white adipocytes into beige adipocytes Download citation DOI: https://doi.org/10.1038/s41568-024-00752-0 Issues with signing in? Click here Need help signing in? A look at how notable co-investment funds from Hamilton Lane HarbourVest and Adams Street have fared recently Your email address is already registered with us. Don't have an account? A verification email is on its way to you. Please check your spam or junk folder just in case. Not for publication, email or dissemination Learn how to find and define your niche in this blog get tips on how to find niche products to sell to your target market On this pageWhat is a niche?Why is it important to find your niche?How to find your niche in 6 steps Niche market examples Tips for finding niche products to sell How to make your niche profitable Find your niche FAQStart your online business today. The best way to succeed online is to pick a niche with characteristics that are conducive to success. Because your niche dictates everything from the products you develop to how you communicate with your customers By understanding the specific needs of a niche you can position yourself in the market and earn more sales. In this short guide, learn how to find your niche and decide what products to sell in it—plus, get tips to see if your small business ideas have sufficient demand A niche is a segment of the broader market that has its own unique characteristics to distinguish it Choosing a niche lets you target a more specific audience and offer products and content tailored to their unique needs and problems you could create ceramic planters that appeal to customers who value craftsmanship and sustainability You’d produce planters with unique designs and colors not found in larger retail stores to further attract buyers and build loyalty Choosing a niche helps you build credibility with one audience and dominate a particular corner of the market to ultimately become the go-to business for a specific group of people If someone is looking for a specific work boot or running shoe Say you position your shoe store to be the No You sell durable slip-resistant shoes that offer support all day long You spend money on ads and influencer campaigns targeting nurses you become the go-to spot for nurse shoes and establish your place in the market Finding your niche begins with looking at your passions and skills Let’s say you have a passion for fashion and are good at styling outfits You might enjoy creating or sourcing clothes and working with a stylist for photo shoots You can find a niche in the fashion industry by combining these interests and skills Or perhaps you enjoy cooking and creating healthy recipes. You might enjoy selling cookware and writing recipes for your company’s food blog Finding a niche in the food industry would be easier with these interests and skills The key is finding an area you’re passionate about and have some skills in This will make finding a niche you enjoy working in much easier Once you have an idea of your passions and skills you want to build a big list of niches so you can determine demand Trail running shoes are a segment of the larger industry of footwear and the audience is runners but here are a few strategies to consider: The best way to start brainstorming is to understand what other online retailers are doing Let’s use “vegan shoes” as a starting point Use the Refine results section to filter by type you can find a goldmine of potential angles like faux leather unisex running shoes or vegan pumps Dig until you find an underserved problem you feel you can provide a solution to Keep in mind that even if some brands are already in the space you can still compete by getting even more specific with your audience and building an effective marketing strategy Start looking outward to catch any common pain points you might notice both youth and elderly people have trouble tying laces Maybe you yourself are after a certain style of shoe but no one ships to your country Take note of any problems like these and be the business to serve that niche population with your unique solution Ever notice how when you start typing something into a Google search bar, it shows you suggestions before you finish? These are Google’s most-searched-for related keywords and queries Use them to your advantage to find a niche for your product category You can plug in “best shoes for” to get some ideas You can also use keyword research tools to check search volume and trends This will help you identify the most viable opportunities The web is pretty good at organizing itself into communities based on shared interests Dig through the most active subreddits and listen in on the discussions Subscribe to hashtags on Instagram and TikTok and you’ll find opportunities to narrow down your niche Read more: What To Sell on Shopify: 17 Creative Ideas for 2025 it’s important to ensure a big enough market to support it It’s no use trying to sell to a group of people that’s too small to sustain your business Here are a few ways to determine if there’s a big enough market for your niche: Do market research on your chosen niches to see which have the most potential You can also plug each niche into Google Trends to research the current trend you can see below that demand for the topic “vegan shoes” has seen some decline over the past five years This would be an indication to either continue narrowing your niche to find a segment within vegan shoes trending upward or pivot to another footwear sector altogether Now that you’ve brainstormed a list of potential niches If you’re struggling to narrow down your list Read more: How This Urban Contemporary Gallery Targets Niche Customers There are a few key ways you can validate your niche before you invest too much time or money If there are already a ton of niche businesses selling what you want to sell, that’s not necessarily a bad thing. It could mean there’s a market demand But it also could mean you’ll have a hard time standing out in the crowd. If you do decide to enter a competitive market, you’ll need to have a unique selling proposition See if potential customers would be interested in what you’re selling and willing to pay for it You can also look for online communities related to your niche and see what kinds of conversations are taking place Are people talking about the problem you’re trying to solve that’s a good sign you’ve found a potential niche Crowdfunding platforms like Kickstarter and Indiegogo allow you to generate awareness about products before they’re even developed You can introduce your idea and gain followers before the idea has come to fruition you’ll have an engaged group of target customers ready and waiting for you when you launch It’s important to be up to date with what’s happening in your chosen niche. Resources like Think with Google and Nielsen consumer research will help you understand consumer pain points Set up Google Alerts for related keywords and regularly monitor social media conversations to stay on top of what’s trending The goal is to get feedback from real people about your niche idea This will help you validate (or invalidate) your assumptions and give you a better idea of whether or not there’s a market for what you’re selling Finally, you can validate a product or niche by testing it out. This can be done by starting a blog or a YouTube channel related to your niche, or by creating a landing page to sell a product or service related to your niche If you can get people to sign up or buy what you’re selling that’s a good sign that you’ve found a viable niche Start selling your products on YouTube from Shopify Shopify comes with powerful tools that help you promote and sell products on YouTube The topic of sustainability has become increasingly important Almost any mass-produced product has a niche market of conscious consumers looking for greener alternatives companies might have donated proceeds to a cause but now consumers care how products are made there’s a subculture within green living that cares about ethical supply chains Make sure your environmental claims are backed up you can’t be perfect—that’s not how business works Keep in mind that greenwashing will always backfire Spending growth in the pet industry could reach 7% a year by 2030. The market offers a lot of product opportunities across different types of pets For example, Pup Socks sells personalized socks you can plaster your beloved pet’s picture on and it has hundreds of thousands of website visitors monthly: While dogs and cats are the most common pets there are plenty of households with more unique companions You can tap into any of these niche markets. Even the world’s biggest brands are adopting niche marketing approaches through targeted local campaigns This is because they’re competing with a consumer-driven movement to support local businesses But if you’re only selling online, it can be difficult to establish a local presence. Luckily, there are ways for ecommerce sellers to appeal to locals. Peace Collective with the slogan “Toronto Vs Everybody.” It has since expanded its product line beyond the city and added a charitable component to its business model. If you want to sell locally you could make t-shirts with subculture slogans or even sell souvenirs and city guides. Read more: What Is a Niche Market? 9 Examples in 2025 but has so many niches available to choose from. you might sell climbing treadmills or simulators for climbers to train indoors or plyometric boxes for boxers looking to improve their speed. Westside Barbell is a fitness ecommerce website that operates in this niche The brand offers lots of products for bodybuilders and even coffee—catering to a multibillion-dollar audience ready to get in shape Even though the world has gone digital, there are still people who value handwritten notes and letters—enough to make up a $158 billion market The market caters to groups like business professionals and hobbyists who appreciate the art of writing The brand also constantly rolls out new collaborations and promos to earn sales and build customer loyalty: The men’s grooming and beauty market is exploding, estimated to reach a $115 billion market valuation by 2028 This growth is driven by a cultural shift toward men taking better care of their appearance and the destigmatization of men’s beauty routines. Players in this market can cater to various men’s needs offering items like beard care and skin care solutions or environmentally conscious products. There are a lot of skin care products for men out there, and Bulldog Skincare stands out in this niche with natural ingredients and sustainable packaging The brand has successfully marketed itself as an easy to use Read more: 50+ Best Shopify Stores to Inspire Your Own While you’re brainstorming various niches and products to sell there are a lot of important things to consider Keep in mind that finding a great product is only part of the equation and you’ll also want to understand a product’s demand Here are some ways to look for niche products to sell: Merchants rarely make much on big-ticket items and will earn only 5% to 10% on products like laptops and TVs Where they really make their money is on the accessories and customers are much less price-sensitive about them A buyer might shop for weeks to get the best deal on a TV but wouldn’t think twice about dropping $30 on an HDMI cable from the same place. When you choose a niche with lots of accessories, you’ll enjoy significantly higher profit margins and fewer price-sensitive shoppers It’s amazing how much money passionate hobbyists will spend Mountain bikers will drop hundreds on lightweight accessories to shave a few pounds off their bike and avid fishermen will invest tens of thousands of dollars in boats and related accessories if you can offer a product-based solution to a painful problem you’ll find a captive audience eager to buy This price range is an ecommerce “sweet spot.” It’s large enough to create decent average order value (AOV) and per-order profit informative website—most customers won’t need to speak with someone personally before they buy the ability to drive sales without the assistance of a large customer service or sales team offers massive efficiency savings But if you’re selling products that cost $500 or more many customers will want personalized customer service before pulling out their credit cards If you needed garden equipment, you’d likely head down to your local Home Depot or Lowe’s. But where would you go to buy surveillance equipment or magicians accessories? Probably online. Picking niche products that are hard to find locally will send your target audience to the web, where they can find your online store While you ideally want something difficult to source locally you also need to ensure there’s ample demand for the product If your product line is constantly changing year to year you’ll end up spending valuable time on resources that will soon be outdated Selling a product line with limited turnover ensures you can invest in an information-rich website that will be viable for years Repeat customers are essential to any business and it’s significantly easier to sell to existing customers who trust you than to new prospects If your product needs to be re-ordered regularly—and you’re able to keep your customers happy—you’ll be on your way to building a profitable business with recurring revenue Having a niche business makes it easier to find potential customers and convince them to buy from you, but you need to be sure there are enough buyers in that niche to make it viable. Getting to profitability involves a few strategic steps to capitalize on the unique elements of your audience: Even if you do achieve success early on, niches change, so expect to evolve with your audience over time. You might even introduce more products to your line as new opportunities present themselves. Understanding the specific needs of each business niche makes it possible to speak directly to them in your marketing efforts—you’ll have a greater chance of attracting a buyer’s attention and winning their business by making it clear that your product is specifically for them. A profitable niche has paying customers with a clear problem they need solved. Look for people already spending money on similar services or products, and test demand by offering something small—like a paid consultation or digital product—to see if buyers show up. Absolutely. Many people refine or pivot their niche as they learn more about what works and what they enjoy. The key is to start with something specific, get traction, and adjust based on real-world experience. It depends on your effort and strategy, but most freelancers and business owners see clarity within a few months and real momentum within a year. The more you test, tweak, and engage with your audience, the faster you’ll find your footing. Finding the right niche means choosing the best target market to sell to. When starting a business, you want to become the go-to seller in a niche to build trust with potential buyers and earn more sales. To find your career niche, start by assessing your skills, interests, and values, then research industries and roles where these align. You can also gain valuable insight and opportunities by networking with professionals in your field and doing internships or projects. Every product is carefully selected by our editors. If you buy from a link, we may earn a commission. Learn more Apple introduced a new iPhone app to little or no fanfare But there’s a reason for its quiet release — it isn’t meant for everybody The app is called Maps Surveyor and its sole purpose to improve one of Apple’s most popular apps: Apple Maps As first spotted by MacRumors Maps Surveyor is an app that’s really only designed for people committed to making Maps Here’s how the Apple Store describes it: “Surveyor helps Apple improve Maps by collecting data such as images of street signs and other roadside details But even though the app is free and available to anybody with an iPhone that doesn’t mean everyone can use it the app prompts you to open another app called Premise Premise is a task marketplace that rewards users with money for sharing information Apple uses Premise to invite specific people it has chosen to help it improve Apple Maps — but that’s reserved for a small group of individuals Even though you can find Maps Surveyor in the App Store the app doesn’t appear to be public-facing at the time of writing Huntington launched Lift Local in October 2020 with a $25 million lending budget It folded the program into the ambitious five-year $40 billion community development strategy it unveiled in June 2021 boosting its lending threshold to $100 million Huntington disclosed Lift Local has made nearly 1,900 Small Business Administration loans totaling $133 million. And while Huntington has made good on more than 75% of the community strategy's $40 billion overall commitment there appear to be no plans to wind down Lift Local "We're continuing to invest in the number of people that are working on the program and some of the technology and protocols we put in place to support it," Small Business and SBA Director Maggie Ference "We're increasing the number of community outreach events we do." Maggie Ference Huntington aims Lift Local at underserved communities veterans and residents in low- and moderate-income neighborhoods about 40% of Lift Local loan dollars have gone to Black and Hispanic entrepreneurs Women and veteran entrepreneurs received an equivalent amount Huntington approved about 7,600 7(a) loans for $1.53 billion Huntington's average 7(a) loan size in fiscal 2024 was $202,000 The average size for Lift Local loans was approximately $71,000. SBA provides guarantees ranging from 50% to 85% on loans made by banks and other private-sector lenders the Consumer Bankers Association awarded Huntington its Joe Belew Award for its work with Lift Local. how do we find ways to be more to the small-business community," Ference said. Lift Local offers loans of $1,000 to $150,000 borrowers receive a bundle of other services accounting and other back office support services. We're going to offer a banker … a champion in the branch that you can come and talk to any time that's going to know what's going on in your business," Ference said "And we're going to give you really patient capital at a low rate along with all these other products and services." the combination of credit and a broad-based support effort appears to be producing solid results Lift Local credit losses "continue to outperform our expectations," Ference said. "When we originally wrote the business plans we were looking at traditional credit metrics that would tell us this is a space that banks shouldn't be lending in," Ference said given the credit scores of some of the participants." 2024 3:33 PM Huntington's experience with Lift Local serves as a hint that current bank underwriting is painting an inaccurate picture of the creditworthiness of many minority borrowers an economist and investment analyst who specializes in impact investing "What it's telling you is that credit policy for small-business loans has been out-of-whack for 30 years," Cunningham said "Small adjustments in the lending process can have an outsize impact on performance Banks need to take a deeper look at the people applying for credit a deeper look at the credit procedure itself." Huntington's ultimate goal is to move businesses past the need for Lift Local and similar programs "Once we actually help them through that process … it helps legitimize them in a way that they don't need the program," Ference said "They're already scoring at a level that they could go to any bank in the country and continue to grow What we love is that they keep doing it at Huntington." An overall winner will be announced at American Banker's Digital Banking event on June 2 Student loans CFPB wins rare judgment over student loan debt relief firm A federal judge has ordered FDATR a now-defunct student loan debt relief provider to pay $43 million in restitution and fees bucking the trend of cases brought by the Biden administration-era Consumer Financial Protection Bureau being dropped Industry News How Cathinka Wahlstrom is modernizing America's oldest bank BNY's chief commercial officer talks about AI tariffs and her efforts to help create a leaner which he thinks reinforces the case for deregulation FORECLOSURE WARS She stopped paying her mortgage more than 15 years ago As low-income households face the dual burden of weather extremes and high energy costs energy efficiency is an increasingly important strategy for both climate mitigation and lower utility bills Passive House standards — which create a building envelope so tight that central heating and cooling systems may not be needed at all — promise to dramatically slash energy costs and are starting to appear in “stretch codes” for buildings And while some builders are balking at the initial up-front cost other developers are embracing passive house metrics as a solution for affordable multifamily housing high performing buildings that are healthy and low energy mainstream everywhere,” said Katrin Klingenberg co-founder and executive director of Passive House Institute-U.S. Klingenberg says the additional work needed to meet an aggressive efficiency standard, is, in the long run, not that expensive. Constructing a building to passive standards is initially only about 3%-5% more expensive than building a conventional single family home or 0%-3% more for multifamily construction “This is not rocket science… We’re just beefing up the envelope We’re downsizing the [heating and cooling] system and now we need someone to optimize that process,” Klingenberg said A Phius-certified building does not employ a conventional central heating and cooling system it depends on an air-tight building envelope highly efficient ventilation and strategically positioned high-performance windows to exploit solar gain during both winter and summer and maximize indoor comfort The tight envelope for Phius buildings regulates indoor air temperature which can be a literal lifesaver when power outages occur during extreme heat waves or cold snaps founder and principal of architecture firm Farr Associates Farr pointed to the example of the Academy for Global Citizenship in Chicago “There was a really cold snap in January Somehow the power went out [and the building] was without electricity for two or three days And the internal temperature in the building dropped two degrees over three days.” Farr said that example shows a clear benefit to high efficiency that justifies the cost “You talk about the ultimate resilience where you’re not going to die in a power outage either in the summer or the winter There is also a business case to be made for implementing Phius and other sustainability metrics into residential construction such as lowered bills that can appeal to market-rate buyers and renters and reduced long-term maintenance costs for building owners founder and CEO of 548 Enterprise in Chicago says in response to questions about how to convince developers to consider factors beyond the bottom line he touts lower operating costs for energy-efficiency metrics rather than climate mitigation when he pitches his projects to his colleagues “I can’t sell people on climate change anymore,” he said the good Lord will catch you when He catch you “But if I can sell you on lowering your operating expenses if I can sell you on the marketability on the fact that your tenants will have 30% that’s a marketing angle from a developer owner that’s what I push on my contemporaries,” Patton said and if your construction costs are not more significant than mine Phius principles can require specialized materials and building practices But practitioners are working toward finding ways to manage costs by sourcing domestically available materials rather than relying on imports “The more experienced an architect [or developer] gets they understand that they can replace these specialized components with more generic materials and you can get the same effect,” Klingenberg said Patton is presently incorporating Phius principles as the lead developer for 3831 W Chicago Avenue, a mixed use development located on Chicago’s West Side. The project, billed as the largest passive house design project in the city to date incorporating approximately 60 mixed-income residential units and 9,000 sq ft of commercial and community space Another project, Sendero Verde located in the East Harlem neighborhood of New York City is the largest certified passive-house building in the United States with 709 units Sendero Verde is designed to provide cool conditions in the summer and warmth during the winter — a vast improvement for the low-income and formerly unhoused individuals and families who live there Even without large upfront building cost premiums and with the increased impact of economies of scale many developers still feel constrained to cut costs “There’s entire segments of the development spectrum in housing where if you’re a developer of rental housing time and again … they feel like they have no choice but to keep things as the construction as cheap as possible because their competitors all do some architecture firms only work with those ‘powerless’ developers and they get code-compliant buildings.” But subsidies, such as federal low income housing credits IRS tax breaks and resources from the Department of Energy also provide a means for developers to square the circle especially for projects aimed toward very low-income residents Nonetheless, making the numbers work often requires taking a long-term view of development principal at Sweetgrass Design Studio in Minnesota Nowak was the designer for Hillcrest Village an affordable housing development in Northfield that does not utilize Phius building metrics but does incorporate net-zero energy usage standards energy-efficient housing,” he told the Energy News Network in June 2023 It affects the employers at Northfield having people that are readily available to come in and fill the jobs that are needed “That’s a significant long-term benefit of a project like this And that is not just your monthly rents on the building; it’s the cost of the utilities as well When those utilities include your electricity and your heating and cooling that’s a really big deal.” Developers like Patton are determined to incorporate sustainability metrics into affordable housing and commercial developments both because it’s good business and because it’s the right thing to do I’m not going to design the greatest architectural building I’m not even interested in hiring those type of architects “I had a lived experience of having my heat cut off in the middle of winter I don’t want that to ever happen to anybody I know ever again,” Patton said “So if I can lower somebody’s cost of living And there’s been a boatload of buy-in from that because those are historically [not] things [present] in the communities I invest in.” It was a moment he will not forget: as a young software developer Mauricio Aniche was team lead for an American company he developed petrol station management software for Central America he travelled to the Dominican Republic to see it in action “We were celebrating the successful launch on the beach when in the middle of the night the software crashed It had not been properly tested and that meant no petrol coming out of the nozzles at petrol stations.” It was a revelation for Aniche “It is also what drew me to becoming a researcher so we could learn how to do things better.” Aniche joined TU Delft in 2016 as a postdoc working in Professor Arie van Deursen’s software engineering department; in 2018 He worked alongside his MSc and PhD studies and he’s always liked having a foot in both camps “I taught master courses and did thesis supervision but my most popular course is the bachelor course in software testing It is part of the first year of the computer science programme and now typically draws some 500 students.” Software Quality and Testing: “99 percent of our graduates who become software engineers will have to solve problems right now That is why I first teach them the state of the practice and then the state of the art” “So a lot of my course is showing students how to make the best use of what we know Later on you can start thinking of how to advance the field.” A bit less theory and more practical tips: this is what he sees as his contribution to a course which was already “pretty good” before he came a sudden spike in student numbers for computer sciences in 2018 called for more action we would rely a lot on teaching assistants but with so many students that was impossible.” That is when Aniche put forward his Fellowship proposal for an automated feedback tool: “Students have to write their own test cases during exercises and exam I wanted to create a tool that could assess students’ solutions and let them know if they are on the right track or not so they can keep trying to solve the exercise by themselves it only takes an additional ten minutes to add the rubrics the tool can use when I create a new exercise Aniche explains “The beauty of it is that we also use the tool to assess exams the exam results of 500 students await me.” ] the number of complaints about grades is really low The tool can assess the exam questions on the components that are taught during the course “The tool checks if their solution touches on every part of the programme as there could well be a bug in one of those ten lines of programme you are not testing This is also the kind of hint the tool can give: ‘you are not testing some lines…’ Another example is the minimum amount of testing “You can test programmes from many different angles but there are certain tests I want students to come up with Only a solution that tests enough and that has ‘the right tests’ gets a 10.” students get to try their hands at more than 60 exercises or in exam mode where they just get a grade “Students already know the tool once they enter the exam and the exam is close to what they have practiced The tool also removes subjectivity in grading so the number of complaints about grades is really low There are things you can’t assess automatically that would matter in real life during lectures we discuss a lot of real life examples of bad software and why it is bad and that will make them better engineers.” The Fellowship allowed Aniche to hire TA’s to create the tool that was first used during the 2022 course “We learned a lot from actually running it as it was already good the way it was.” The tool so other universities can use it as a TU Delft certified plug-in Nijmegen University will be the first to do so and to Martin Mladenov and all the students that helped me develop Andy.” While Aniche is very happy with Andy as a tool for formative and summative assessments he would like to see another shift in the way students are graded “There is too much emphasis on grades alone and this puts lots of pressure on students so they will focus too much on their exams but we should do so in a way that they will concentrate on learning That is something we should discuss in the Teaching Lab.” Now employed as Tech Lead at payment platform Adyen Aniche plans to return every year to teach this particular course While he misses the inspiration he got from his students and being free to explore his own research ideas he still has his hand in teaching in a way “I am involved in upskilling engineers in the sense of lifelong learning We also try to improve the on-boarding experience because of the complexity of the software systems” he says. He would like to bridge the gap between graduation and being able to work in industry like three-month boot camps to ready our students for their postgraduate career There is so much students need to know if they want to work in industry like Spring Boot or PostgreSQL – the tools that most companies use and that will support them throughout their careers that will enable them to learn technologies that don’t exist today with ease But that doesn’t count on day one of your career in industry.” In the latest chapter of their nearly decade-long rivalry, British artist Stuart Semple takes a bold new step by legally changing his name to Anish Kapoor in a playfully provocative response to the British sculptor’s exclusive rights to Vantablack the infamous ‘world’s blackest black.’ In a newsletter titled I’ve Just Changed My Name Semple—writing under the name Anish Kapoor—informed his followers of his name change stating I legally changed my name to Anish Kapoor today now I can get hold of that Vantablack and share it with you all originally developed by Surrey NanoSystems for military and aerospace applications before Kapoor’s exclusive deal restricted it from the art world A full-color copy of Semple’s official legal name change deed comes with every print celebrating his ongoing satire on accessibility It was a quiet time between the two artists until one of them signed exclusive rights to a paint dubbed the blackest shade of black as it could absorb 99.965% of visible light The company originally developed it for military applications namely satellite-borne blackbody calibration systems but because of the limitations in how it was manufactured they switched to spray-painted Vantablack coatings This shade caught the eye of the sculptor Anish Kapoor who had already produced a hole made of black pigment It was a natural move for the artist to seek a similar shade he responded to Anish Kapoor’s rights to Vantablack by making his own paint named Blink He describes it as the ‘blackest black ink,’ suitable for pen and ink work There’s a catch here: anyone can purchase and use Blink a series of archival giclée prints hand-signed with Stuart Semple’s newly adopted name there have been four generations or batches of Blink when users try to acquire the paint from Stuart Semple’s shop site Culture Hustle there’s a note saying that the buyer confirmed they are not ‘Anish Kapoor you are in no way affiliated to Anish Kapoor you are not purchasing this item on behalf of Anish Kapoor or an associate of Anish Kapoor this material will not make it’s way into the hands of Anish Kapoor.’ Looking at the packaging design of the upgraded Black 4.0 of Stuart Semple the logo seems to resemble the middle finger that Anish Kapoor dipped into the British artist’s World’s Pinkest Pink powdered paint Even this shade of pink has the disclaimer that anyone who buys it should not be related to and associated with Anish Kapoor The dubbed feud between the artists may reach their decade celebration in 2026 Stuart Semple pulls a surprise for Anish Kapoor by changing his name to his All eyes are now on Anish Kapoor to see if he’ll respond to this stunt—and if their past exchanges are any indication his reply will likely come in artistic form the print will be available for 72 hours starting November 1 boldly emblazoned with the words ‘I’m not a niche man a full-color copy of Semple’s official legal name change deed comes with every print the dubbed feud between the artists may reach their decade celebration in 2026 this artwork takes the ongoing saga between the two artists to a new level of wry humor anyone who buys this shade should not be related to Kapoor | image courtesy of Anish Kapoor via @dirty_corner Kapoor secured exclusive contract rights to the pigment | image courtesy of Anish Kapoor via @dirty_corner Semple pulls a surprise for Kapoor by changing his name to his | image courtesy of Anish Kapoor via @dirty_corner name: I’M NOT A NICHE MAN artist: Stuart Semple | @stuartsemple AXOR presents three bathroom concepts that are not merely places of function but destinations in themselves — sanctuaries of style Login | Sign Up Push Square Guest Login or Sign Up we're going to explain everything you need to know about Carving Out a Niche On this page: Final Fantasy 16: Carving Out a Niche Walkthrough enlists Clive's aid in finding out how to make a carving knife that somehow "embodies the spirit of Dalimil." Carving Out a Niche begins when you speak to Sava Your job this time is to help him craft a carving knife Head to the marked location and speak to the two townspeople Your next stop is a marked spot in the desert Head over there to track down a metal trader Return to Sava and talk to him to end the quest Did you follow this walkthrough for Carving Out a Niche? For more information on Final Fantasy 16, including All Quests, refer to our Final Fantasy 16 guide through the link Stephen has been part of the Push Square team for over six years bringing boundless enthusiasm and a deep knowledge of video games to his role as Assistant Editor Having grown up playing every PlayStation console to date He's also our go-to guy for Sonic-related matters Hold on there, you need to login to post a comment.. Clair Obscur: Expedition 33: The Continent Walkthrough - Lost Gestrals, Music Records, Outfits, Journals All collectibles locations in The Continent Clair Obscur: Expedition 33 Trophy Guide: All Trophies and How to Get the Platinum How to unlock all Trophies in Expedition 33 Clair Obscur: Expedition 33 Guide: A 100% Collectibles Walkthrough Clair Obscur: Expedition 33: All Journals Locations Where to find every Journal in Expedition 33 Clair Obscur: Expedition 33: All Outfits and How to Get Them Where to find every Outfit for all characters Show More © 2025 Hookshot Media, partner of IGN Entertainment | Hosted by 44 Bytes | AdChoices | Do Not Sell My Personal Information This story is part of Retail TouchPoints’ ongoing “Small Business focusing on smaller retail brands that have found big success and have even bigger ambitions although you’d have a hard time telling it from his accent “I was raised in England and according to my staff my Scottish accent only appears when I do two functions in the shop — when I take money or I say goodbye,” he said “I feel deeply culturally worried that this is a genetic thing Whether north or South of Hadrian’s Wall, he’s a legit Brit, which gives him all the street cred he needs for his business as the owner of the San Francisco-based Willow on the Green More often it’s simply referred to as the “British cheese shop” though not only because “Willow on the Green is a very esoteric name,” admits Sinclair we’re the only British-dedicated cheese shop in America,” he said So how does one get into such a niche area of retail In the process we learned a lot about cheese but also how thoughtful curation has helped him create a thriving local business in a country where his product is largely unknown (despite being a former British colony). Retail TouchPoints: Tell us how the shop started and where you came up with the name Alex Sinclair: I started the business in the middle of COVID Normally I go home every other year and get my fix of Michelin food and fancy stuff that you can’t get in this country so I basically started my own shop so I could get access to what I would have at home and I figured cheese and bread together would work Willow trees make picnic baskets and it’s also the basket that’s used to mold cheese in so Willow on the Green basically means “picnic on the park.” The funny thing is that because I have Scottish roots many people call it Willow on the Glen to the level that I had to buy the domain so it forwards back over to me but are the Brits really known for their cheese One typically thinks more of France or Italy in that regard Sinclair: Well France had good billing by the Roman Empire but we Brits have been producing cheese for about 5,000 years most of the early cheeses that we created were goat and sheep because that’s what you have in the UK Essentially cheddar was taught to us via Rome, and that’s when Cheddar Gorge [in Somerset But then after the World War and with the ’80s corporate greed type of thing creameries took over a lot of the traditional makers and marginalized and almost killed our industry We went from having five creameries making 10 cheeses — and killing all of our history — to 870 varieties That’s where it becomes very interesting because now our industry is mostly making cheese for Brits since [other countries have made a name for themselves in cheese] We ended up in a scenario where it’s like well you don’t really want our stuff: Wensleydale sounds funny Stilton is an upper crust thing and Cheddar is default but because we never protected the word it can be used in this country RTP: So how are you bucking that stereotype with your shop Sinclair: One factor is that because Brits are historically and genetically lactose intolerant to specific breeds over centuries we’ve developed farming practices to cope with it but that’s because our farming practices don’t allow for things like meat-producing cow breeds to be mixed with milk-producing cow breeds we don’t feed them allergens and they’re generally grass fed Part of the reason this shop came about is because I lived in America for a while and then I ended up in Japan for about five years I just kind accepted it and [decided I had] developed an allergy I live in a neighborhood in San Francisco that is traditionally Southeast-Asian Pacific and Western European both [cultures] that don’t want to eat anything that will upset them They’ll eat European cheese as a luxury but not every day But if you put British cheese in front of them and say “You don’t need to have an enzyme capsule,” and they have it and they’re perfectly fine So we designed the shop to be very specifically focused on British cheese but those are mainly goat- and sheep-focused because goat cheese is 99% nonreactive But it’s better to put a British flag over the shop than say “We’re the kings of lactose intolerance-supportive cheese.” and it’s not because we’re trying to make sales but it’s more of an education process You have to train a whole generation and a whole neighborhood and city about A what cheeses [lactose-intolerant shoppers] can actually have RTP: What else does your shop carry beyond cheese and what are you looking for in terms of the product assortment Sinclair: Just the same way as I go farm to farm curating cheeses I’m going to small manufacturers and independent makers in the UK and bringing those products into America for the very first time There’s a company called Olives Et Al that brines its olives in olive oil There’s Puckett’s Pickles by Sarah Puckett; she does condiments very We also work with PekoeTea in Edinburgh and Isle of Skye salt — that’s a couple that hand-rakes sea salt from the Isle of Skye if you were in London you would find in the most luxury-oriented stores [It’s great for the brands too because] we promote them hitting California for the first time It becomes a feedback loop where locals in the UK will go very fancy people in California love this thing We also just got our beer and wine license — it took eight months to get it through the San Francisco system So now we’ve got British sparkling wines if you’ve got cheese you need crackers and biscuits; if you’ve got biscuits but if I don’t keep my customers always engaged and excited for what is coming then I have no way to keep them in my store and to spread the word out about what the shop is You can go to Trader Joe’s or World Market and grab cheap British product very easily I think the difference between my shop and other British businesses is that the focus in this country tends to be on selling candy or sweets [When that’s the case] you’re not selling the best of British you’re selling what you would get in a corner shop or a lower-grade grocery store things that are commodity level but sell for an extended profit here And that’s not really what I’m going for I find products on [B2B marketplace] Faire I also have seven British distributor-importers that I work with for other non-exclusive Then I have three cheese distributor-importers I work with sometimes four or five if I’m pushing into the rarer stuff And right now I have four alcohol distribution importers We’re a 350-square-foot shop — all that business just to have this small niche It’s about 1,600 SKUs but they’re not always sitting there at all times My MBA thesis was linked to subscription video-on-demand systems and tactics Max — it’s all about how you keep someone engaged through surprise and interest RTP: You refer to yourself as a cheesemonger Does that require any special certification or training cheesemonger in America comes in different flavors you have developers that work with cheese makers and then you have people who do work treating cheeses There’s the American Cheese Society that does some certification but the concept of cheesemonger in America has always been are you someone who owns a cheese shop or works with a cheese shop; someone who’s going farm to farm and having conversations with cheese makers and developing those relationships to understand the quality and filtering out badly made product so that when you present it to a customer it’s the highest quality That’s really what I’m saying [when I call myself a cheesemonger] — I’m doing the editing and curation process rather than you just going to a supermarket and being shoved in an aisle of random cheeses that are generally machine block-made and have no “terroir” to them whatsoever I can tell you precisely the county this was made in and the farmer that made it [things like] this is definitely a Stinking Bishop and I’ve made sure it’s gone through all of the right processes Sinclair: Oh yeah, Charles Martell makes fabulous cheeses that are godly expensive He has his own orchard where he makes pear cider [“perry”] that he washes the cheese in you get a mini one for Christmas or if you’re in a luxury house in Somerset they’re cutting it up on a very expensive cheese board we’re not really in the business of need View the Retail Trendcaster Webinar Series on-demand to uncover key 2025 retail trends from AI and personalization to social commerce and actionable takeaways to stay ahead in a rapidly evolving market Get access to exclusive content including newsletters with experts analyzing holiday sales and forecasting trends View the full lineup of the Retail Trendcaster video series for insights on consumer spending and more—helping you focus on what truly matters in 2025 (Photo by Epics/Getty Images) Crypto would be better off remaining a niche The greatest crisis in crypto so far has been the rapid decline and tremendous fall of FTX At the time of the collapse of what turned out to be Sam Bankman-Fried’s personal piggy bank Its demise caused shockwaves across the industry bringing down not just prices but a litany of companies This article is excerpted from The Node, CoinDesk's daily roundup of the most pivotal stories in blockchain and crypto news. You can subscribe to get the full newsletter here it was unclear if crypto as a concept would ever recover – the blatant fraud of what was among the most consumer-savvy and trusted crypto companies appeared to confirm the widespread assumption that all of this was just artifice covering up fraud the cyclical nature of the market has been an accepted part of life But isn’t it odd that this maturing industry has normalized these boom-and-bust cycles It seems to me that mass adoption for any blockchain or consumer application is contingent upon the price of its token – or the industry itself – not always being at risk of imminent collapse See also: You Want Crypto Regulation? I’ll Give You Crypto Regulation | Opinion the biggest problem with growing crypto is the growth of crypto This whiplash between euphoria when the markets surge and despair when it shrinks is a result of crypto’s pursuit of mass adoption the things that fail are the things that blockchains were built to mitigate or replace were built to make crypto palatable and/or easy-to-use It’s not an uncommon opinion that “the masses” likely won’t self-custody what’s even the point of something like Bitcoin “The risk of growing adoption is that new entrants aren’t aware of Bitcoin’s core principles: decentralization the features that make them reality may not remain in the protocols over time,” said Alex Thorn the head of firmwide research at investment bank Galaxy Digital See also: An Ode to LocalBitcoins | Opinion Adoption means following the law (which is often at odds with crypto's values) and creating easy-to-use sign-ins and on-ramps (which can be compromised) There is a tension – if not direct competition – between the aims of decentralization and mass adoption and you risk destroying what it is actually useful for “Simply becoming folded into the dominant financial system ends up ceding a lot of the opportunities that matter with this tech,” said Nathan Schnieder professor of media studies at the University of Colorado Boulder and author of “Governable Spaces.” It’s a point echoed by University College Dublin lecturer Paul Dylan-Ennis who said “crypto is a subculture that cannot accept it is a subculture Most of our troubles stem from how talk of 'onboarding the next billion' causes us to decay our values.” There all alongThere is a certain irony that developers founders and investors have spent 15 years and billions of dollars searching for a “killer app” for blockchain and those who actually walk in his footsteps have built digital bearer instruments that can be used any which way and cannot (easily) be taken from you as in other periods where it seems like crypto is right on the cusp of breaking through this usage pales in comparison to the speculative use of crypto jumps around from coin to coin or protocol to protocol and causes the number to go up – essentially creating a circular economy Gambling is a use case to a certain extent But if people want crypto to be used productively founders and investors should be building for people who have an actual need for censorship-resistant money and tools author of crypto-critical news services Web3IsGoingGreat and “Citation Needed,” argues that crypto is already mainstream “There are individual projects that are still small and niche but with Brian Armstrong and Sam Bankman-Fried rubbing elbows in Congress and BlackRock and Fidelity launching bitcoin ETFs I think that ship has probably sailed,” she said in a direct message educator and monero superuser SethforPrivacy sees things differently. The “unfortunate reality is that most people don't yet realize the need for Bitcoin nor are they willing to take on that much personal responsibility and as such we must focus our efforts on improving Bitcoin for those who do realize the need today," he said See also: In Defense of Meme Coins | Opinion There’s also an argument that decentralization is precisely the reason crypto will go global “The ONLY thing that makes Bitcoin’s global ascension possible is its most cypherpunk attribute: that it is owned by no one not states or corporations," said Alex Gladstein chief strategy officer at the Human Rights Foundation it’s not exactly clear what the masses want admits that “while we believe *everyone* wants privacy censorship-resistance and protection against nation-state attacks some people are fine with a product that solves a problem and has good UX.” censorship resistance and maximum decentralization “We should explore getting crypto in the hands of as many people as possible." Likewise, Roko Mijic, of “Roko’s basilisk” fame, argued that it’s actually scale that gives decentralized tools any power, which is observably true in that Bitcoin is difficult to attack because it has miners spread across the world. “You can't resist censorship from inside a small-scale crypto network because the government will just bring down the whole network,” Mijic said. Justin Ehrenhofer, founder of Moonstone Research in Chicago, echoed this sentiment, pointing out that a currency is only useful if it is widely accepted, and so “cypherpunks should focus on building systems that appeal to outsiders.” However, he did add that “with wide-scale adoption” there has been a degradation in the spirit of crypto, given that the average user stores their wealth in custodial exchanges. I suppose the question is, how valuable are crypto’s core values? Note: The views expressed in this column are those of the author and do not necessarily reflect those of CoinDesk, Inc. or its owners and affiliates. Most people don’t. It’s just that I happen to live in Manhattan, and below 14th street, it’s safe to expect on any given day that at least one person will come up to me and say hello. I’m a Niche Internet Micro-Celeb. I read that on Reddit. I search my name on there multiple times a week, even though a new comment about me pops up only about once a month. I hit on a male model at a Christmas Party last year. I didn’t exactly hit on him, actually—I did something a little more to the point: I walked on up and told him to come home with me. He lowered his voice: “Aren’t you, like, that Carrie Bradshaw girl?” He said it with such malice that I went home alone and banged my head against the wall enough times to forget his existence forevermore. But he made a fair point. I overshare, publicly and professionally. I get paid to write about my exploits. I used to want everyone in the world to fall in love with me, but now I just want everyone in the world to pay attention to what I have to say. Most of what I have to say is about sex. I talk about sex and I talk about love and I talk about getting rejected. (Nobody wants to hear about the times when the sex is good, anyway.) Men give my life meaning. I might be the straightest woman on planet earth. I see the world and make sense of my personhood by documenting my romantic endeavors. The difference between my dating life now, in 2023, from two years ago—before I was getting paid to write—trips me the hell out. I didn’t plan on becoming some sort of botched Bradshaw. My childhood heroes wouldn’t understand me at all. When I feel irreparably tacky, I remind myself that the artists I admire probably weren’t respected by their elders, either. I’m too outspoken and angry to be as edible as Carrie, anyway. I took a middle-aged father home from a film party and was surprised to learn he already knew who I was (no fathers should know who I am) (if you’re a father, get the hell off my Twitter). We drowned ourselves in a makeout session for awhile, and I thought about calling him Daddy. But it ended when he stopped himself from putting his hand down my pants. He looked at me fearfully. “Please don’t write about me.” He really did look scared. As my twenties neared a close, I got tired of chasing. I wanted a boyfriend who would make my whole life better. I soon fell in love, cheated on him mercilessly, and ended up breaking up with him a year and a half later. I thought I’d find a better one. I did not. I wound up single for four treacherous years. Until now. I met him in February. I’m not single anymore. I have a boyfriend. I finally did it; I got a boyfriend. The four years of singledom make sense to me now: I hurt myself and I begged and I fought and I lost, all so that I could be ready for him. We kissed on the Upper West side, outside the fancy Chase bank on 72nd street, one blustery February night, and I knew I would have to grow up. Cliches have become the only way to explain the reciprocity and trust and danger and adventure that I feel with him. A girl came up to me on the street early one September morning and congratulated me on getting a boyfriend. I was surprised she knew about him—I hadn’t so much as tweeted a reference to a boyfriend. I’d been careful. The girl told me she lives on the same block as me—she might have said two doors down—and that she saw us together every morning. “He’s hot,” she said—a vocal high-five. “I feel like if you can do it, so can I.” My schtick is that I’m an underdog. Or maybe it’s not a schtick, maybe that’s just my position in life. Some people root for me because I’m messy and don’t have the good sense to hold back. I’ve learned that the shortcut to connection is to let it all spill out. Maybe not for everyone, but for me. It’s a trick that recently I’ve learned is simply that … a trick. I’m going to have to live with being vague. Metrics details Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs) The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance Although leukemic infiltration of the spleen is a hallmark of CML it is unknown whether spleen cells form a niche that maintains LSCs we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs) These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state resulting in disease progression and resistance to therapy whether spleen cells form a niche and regulate LSCs has not been studied so far we characterized the splenic leukemia stem and progenitor cell (LSPC) niche in CML We found that the spleen harbored predominantly primitive LSCs which were quiescent and more resistant to imatinib treatment compared to LSCs from the BM leukemic cell differentiation in the spleen was impaired leading to an accumulation of common myeloid progenitors (CMPs) Gene expression analysis of LSPCs revealed an enrichment of stemness genes and a downregulation of genes involved in myeloid lineage commitment in the spleen LSCs in the spleen exclusively located in the red pulp and were dependent on RPMs Splenectomy reduced the accumulation of LSCs and prolonged survival of CML mice RPMs in the spleen contribute to disease progression and resistance to TKI therapy in CML by accumulating a large pool of primitive LSCs All experiments were performed with sex- and age- matched (6–12 weeks) animals according to Swiss laws for animal protection No randomization was used for animal studies Splenectomy was performed as previously published [16] and animals were allowed to recover for at least 2 weeks before CML induction Lineage depletion was performed using biotinylated antibodies against red cell precursors (anti-Ter119) magnetic activated cell sorting (MACS) anti-biotin beads and an autoMACS pro seperator (Milteny Biotec) Negative and positive separations were stained with fluorescent antibodies and analyzed on a BD LSR Fortessa (BD Biosciences) device or sorted with a BD FACSAria (BD Biosciences) Data were analyzed using FlowJo software (TreeStar) BM and spleen were analyzed 18 days after transplantation Endpoint in survival experiments was determined as day 60 after transplantation 5 × 104 LSCs from BM or spleen were transplanted into non-irradiated or sublethally (4.5 Gy) irradiated BL/6 recipient mice LSC frequencies were determined by transplanting 1 × 106 and 1 × 104 FACS-purified GFP+ cells into lethally irradiated (2 × 6.5 Gy) BL/6 recipient mice BL/6 CML mice were treated with 1 ml of clodronate liposomes (equal 5 mg of clodronate) (Liposoma the Netherlands (clodronateliposomes.com)) or vehicle (PBS) every 5th day by intraperitoneal injection starting 3 days prior to CML induction Residual contaminating lymphocytes were gated before clustering based on their CD19 and CD3 expression P values < 0.05 were considered significant All animal experiments were approved by the veterinary office of the Canton of Bern Analysis of human samples was approved by the local ethical committee of the Canton of Bern This suggests that CML is first initiated in the BM and LSPCs migrate to and accumulate in the spleen during disease progression spleen and blood of BL/6 CML mice 18 days after transplantation One representative experiment out of 5–10 is shown B Limiting dilution transplantation of BCR-ABL1-GFP+ cells from spleen or BM into lethally irradiated BL/6 mice C–G Frequencies of LSPC subsets in spleen and BM 18 days after CML induction H Kaplan–Meier survival curves of secondary BL/6 CML mice 2 × 104 FACS purified LSCs from spleen or BM were transplanted into sublethally irradiated BL/6 recipient mice Data are pooled from two independent experiments with n = 13–15 mice per group I–J Frequencies of L-progenitor cells in spleen and BM 18 days after CML induction 5 × 104 FACS purified LSCs from BM or spleen were secondarily transplanted into non-irradiated BL/6 mice LSPCs in BM and spleen of secondary recipients were analyzed 24 days after transplantation The ratio of the frequency of the individual cell population in spleen versus BM is indicated Pooled data from 2 independent experiments with n = 5–10 mice per group is shown The frequency of L-CMPs was increased in the spleen with a concomitant decrease of the more differentiated L-GMPs (Fig. 1I, J, Fig S2L) these results demonstrate that the spleen harbors greater numbers of functionally competent primitive LSCs than the BM with an impaired differentiation of leukemic progenitor cells These results indicate that the splenic microenvironment differentially affects LSPC accumulation and differentiation compared to the BM microenvironment BL/6 mice were splenectomized or sham-operated 14 days prior to CML induction A Numbers of BCR-ABL1-GFP+ granulocytes/µL blood measured at day 14 and 18 after CML induction B Kaplan–Meier survival curve of primary transplanted splenectomized or sham-operated BL/6 recipients Pooled data from three independent experiments with n = 11–15 mice per group are shown C–G Absolute numbers of different leukemic cell subsets in the BM of splenectomized and sham-operated animals at day 18 after CML induction Pooled data from 3 independent experiments with n = 13 mice per group are shown B Heatmap visualizing the expression of the significantly differentially expressed genes in LSCs and L-CMPs Rows and columns were grouped using unsupervised hierarchical clustering C Expression of genes involved in myeloid expression in LT- and ST-LSCs from BM and spleen Data were clustered using standard Euclidean’s method based on the average linkage and heatmaps were generated according to the standard normal distribution of the values D Gene set enrichment analysis (GSEA) representing the normalized enrichment score (NES) and false discovery rate score (FDR) of gene sets linked to stemness and myeloid differentiation for LSCs (spleen vs E 103 FACS-purified L-CMPs from spleen and BM of CML mice were plated into methylcellulose and colonies were counted 7 days later One representative experiment out of 3 is shown with n = 6 mice per group F LSCs and L-CMPs from spleen were co-cultured for 36 h with spleen and BM flush as supernatants Depicted are log2 fold changes in gene expression in LSCs and L-CMP cultured with BM or spleen flush supernatant One representative experiment out of 2 is shown with n = 3 biological replicates our results suggest that the spleen provides a microenvironment that confers stemness and lacks differentiation signals for LSPCs A BrdU incorporation of LSCs in the spleen and BM 18 days after CML induction and 12 h after intraperitoneal BrdU administration One representative of 4 independent experiments with n = 4–6 mice per group is shown B Frequency of viable and apoptotic LSCs in the BM and spleen of CML mice 18 days after CML induction One representative out of two independent experiments with n = 5 mice per group is shown BL/6 CML mice were treated with imatinib (100 mg/kg BW) or vehicle (H2O) twice daily by oral gavage starting 4 days after CML induction D Degree of BM infiltration with leukemic cells at day 18 after CML induction E To compare resistance between BM and spleen the spleen:BM-ratio of LSPCs was calculated F Relationship between leukemia load in the BM and the spleen:BM-ratio of LSPCs Straight lines represent linear regression calculations dashed lines represent upper and lower 95% confidence intervals Pooled data from two independent experiments with n = 5–8 mice per group are shown G Leukemia load at day 25 in peripheral blood of lethally irradiated secondary recipient mice that received 2 × 106 whole-BM or -spleen cells from imatinib- or vehicle-treated primary CML mice (n = 3) H Kaplan–Meier survival curve of imatinib-treated CML mice that were splenectomized or sham-operated prior to CML induction our results demonstrate that the microenvironment in the spleen renders LSCs quiescent and resistant to TKI therapy A 3D confocal laser scanning microscopy of the spleen 10 days after CML induction Representative images from 3 independent experiments with n = 6 mice are shown BL/6 CML mice were treated with clodronate liposomes (1 mg) or vehicle (PBS) every 5th day by intraperitoneal injection starting 3 days prior to CML induction Spleen and BM were analyzed 18 days after CML induction C Frequency of RPMs after clodronate treatment Pooled data from 2 to 3 independent experiments with n = 5–13 mice are shown I Incorporation of BrdU in LSCs of the BM and spleen of clodronate or PBS treated CML mice (n = 6–8 mice) 1.5 × 103 FACS-purified LSCs were co-incubated with RPMs for 48 h and subsequently plated into methylcellulose Numbers of colonies were counted after 7 days Pooled data of two independent experiments with n = 5–6 samples per group are shown 1.5 × 103 FACS-purified LSCs were co-incubated overnight with 1 × 104 FACS-purified RPMs in the presence or absence of imatinib and plated into methylcellulose colonies were washed and 1 × 104 cells were plated into methylcellulose One representative experiment with n = 3 biological replicates is shown Spicwt/wt donor LSK cells were transduced with BCR-ABL1-GFP and transplanted into Spicwt/wt and Spic–/– mice The leukemic compartment of the CML mice was analyzed 15 days after CML induction Absolute numbers of (B) total leukemic cells One representative experiment out of 3 is shown with n = 5–6 mice per group F Frequency of L-lin− cells in the spleen and BM I 105 total spleen cells from CML mice were plated into methylcellulose and BCR-ABL1-GFP+ colonies were enumerated after 7 days Fold change (FC) numbers of colonies from Spicwt/wt and Spic−/− spleen cells are shown One representative experiment out of 2 is shown with n = 6 mice per group J Incorporation of BrdU in LSCs of the BM and spleen of Spicwt/wt and Spic–/– CML mice One experiment with n = 8 mice per group is shown K Total leukemic cells in the BM of Spicwt/wt and Spic–/– CML mice Pooled data from 2 independent experiments with n = 10–11 mice per group is shown O 105 total BM cells from CML mice were plated into methylcellulose and BCR-ABL1-GFP+ colonies were enumerated after 7 days FC of numbers of colonies from Spicwt/wt and Spic–/– BM cells are shown One representative experiment out of 2 is shown with n = 5 mice per group our results suggest that leukemic RPMs form a niche that retains LSCs in the spleen contributes to their maintenance and quiescence as well as to disease progression and resistance to therapy A Mean distance between CD68+ macrophages and CD34+ human stem/progenitor cells or CD68−CD34− cells in 4 CML patients B Representative immunohistochemical staining (patient 1) for CD68 (brown) and CD34 (red) Stem/progenitor cells (CD34+ non-vascular cells with a nuclear morphology compatible with immature cells (large nuclei open chromatin)) are annotated with yellow circles the irradiation of the recipient and the TKI studied may influence the number and function of LSCs in the spleen The identification of the cytokines produced by RPMs that induce quiescence in LSCs requires further studies whereas fetal liver GMPs were reduced compared to the BM and similar to what we have found for leukemic differentiation in the spleen gene expression analysis revealed upregulation of myeloid lineage–specific transcripts in CMPs from the BM compared to the fetal liver Our study now indicates that RPMs in the spleen crucially contribute to the resistance of CML LSCs to TKI by maintaining LSC quiescence Right on target: eradicating leukemic stem cells Getting to the stem of chronic myeloid leukaemia Regulation of hematopoietic and leukemic stem cells by the immune system Normal and Leukemic Stem Cell Niches: Insights and Therapeutic Opportunities Hematopoietic stem cell niches produce lineage-instructive signals to control multipotent progenitor differentiation The leukemic stem cell niche: current concepts and therapeutic opportunities Acute myeloid leukemia transforms the bone marrow niche into a leukemia-permissive microenvironment through exosome secretion Inhibition of CXCR4 in CML cells disrupts their interaction with the bone marrow microenvironment and sensitizes them to nilotinib Requirement for CD44 in homing and engraftment of BCR-ABL–expressing leukemic stem cells Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche A perisinusoidal niche for extramedullary haematopoiesis in the spleen Macrophages retain hematopoietic stem cells in the spleen via VCAM-1 Leukemic spleen cells are more potent than bone marrow-derived cells in a transgenic mouse model of CML Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis Survival surgery: removal of the spleen or thymus Metoclopramide treatment blocks CD93-signaling-mediated self-renewal of chronic myeloid leukemia stem cells Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates High‐dimensional single‐cell analysis with mass cytometry CyTOF workflow: Differential discovery in high-throughput high-dimensional cytometry datasets ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia Transcriptional heterogeneity and lineage commitment in myeloid progenitors Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2 axis S100A9 induces differentiation of acute myeloid leukemia cells through TLR4 “Stemness”: transcriptional profiling of embryonic and adult stem cells Transcriptional analysis of quiescent and proliferating CD34+ human hemopoietic cells from normal and chronic myeloid leukemia sources Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1 Recombinant human stem cell factor synergises with GM-CSF IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages Interleukin 6 induces myeloid differentiation of a human biphenotypic leukemic cell line and IL-5 family of cytokines: regulators of inflammation Regulation of normal and leukemic stem cells through cytokine signaling and the microenvironment Cytokines regulating hematopoietic stem cell function Role of SCF-expressing bone marrow populations in hematopoietic and leukemic stem cell regulation TNF-α-induced bone marrow stromal progenitor alterations enhance leukemic stem cell growth and treatment resistance via increased CXCL1-CXCR2 signaling CCL4 enhances preosteoclast migration and its receptor CCR5 downregulation by RANKL promotes osteoclastogenesis TNFα coordinates hematopoietic stem cell survival and myeloidregeneration A novel function of interleukin-10 promoting self-renewal of hematopoietic stem cells Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro CD34+ progenitors are resistant to apoptosis induced by a wide range of proapoptotic stimuli Targeting quiescent leukemic stem cells using second generation autophagy inhibitors Treating the chronic-phase chronic myeloid leukemia patient: which TKI Functions and development of red pulp macrophages Hendrikx E Liposomes for specific depletion of macrophages from organs and tissues The stem cell niche in health and leukemic disease Predicting complete cytogenetic response and subsequent progression-free survival in 2060 patients with CML on imatinib treatment: the EUTOS score Response of chronic myelogenous leukemia patients to COAP-splenectomy Intensive combination chemotherapy (ROAP 10) and splenectomy in the management of chronic myelogenous leukemia Splenectomy in chronic myeloid leukemia and myelofibrosis with myeloid metaplasia Mesenchymal niche-specific expression of Cxcl12 controls quiescence of treatment-resistant leukemia stem cells Radiation and the microenvironment—Tumorigenesis and therapy Low-dose irradiation programs macrophage differentiation to an iNOS+/M1 phenotype that orchestrates effective T cell immunotherapy Devastation of adult stem cell pools by irradiation precedes collapse of trabecular bone quality and quantity Effects of irradiation on cytokine production in a mouse model of Behçet’s disease Efficacy of tyrosine kinase inhibitors on a mouse chronic myeloid leukemia model and chronic myeloid leukemia stem cells Role of tumor-associated macrophages in hematological malignancies Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells Colony-stimulating factor-1 receptor is required for nurse-like cell survival in chronic lymphocytic leukemia CSF1R inhibitors exhibit antitumor activity in acute myeloid leukemia by blocking paracrine signals from support cells Tumor-Associated Macrophages in Hematologic Malignancies: New Insights and Targeted Therapies Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin—SCL/TAL1—CD44 axis Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling Download references This work was supported by grants from the Swiss National Science Foundation (31003A_149768 and 310030B_13313 to A.F.O and the Swiss Cancer League (KLS-3346-02-2014 to AFO) EDB and MAA were supported by a MD-PhD scholarship from the Swiss Cancer League and the Swiss National Science Foundation EDB and MAA were further supported by the Ursula Hecht Foundation CMS was supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation (P300PB_171189 and an International Award for Research in Leukemia from the Lady Tata Memorial Trust SSB was supported by a Stanford Bioengineering Clark Fellowship and a Stanford Bio-X Interdisciplinary Graduate Fellowship We thank Ursina Lüthi for excellent technical assistance and the Translational Research Unit Open access funding provided by University of Bern These authors contributed equally: Elias D Bührer Graduate School of Cellular and Biomedical Sciences Department of Medical Oncology and Hematology University Hospital and University of Zürich Stephan Isringhausen & César Nombela-Arrieta Department of Pathology and Neuropathology University Hospital and Comprehensive Cancer Center Tübingen EDB and MAA designed and performed experiments SF performed experiments and analyzed data SI and CNA performed and analyzed laser scanning microscopy experiments JZ and DS performed CyTOF experiments and analyzed data CR designed experiments and interpreted data All authors revised the manuscript and approved its final version Download citation DOI: https://doi.org/10.1038/s41375-022-01682-2 Experimental & Molecular Medicine (2024) Signal Transduction and Targeted Therapy (2024) « Back “It was all gas and no breaks,” says Angelica Aranda ’23 of her frame of mind when she decided not to pursue law school She was going to pursue art—even while realizing that “funding yourself as an artist is not easy.” But that didn’t stop Aranda from figuring out how to push forward politics and law seemed like a natural progression for someone conscious about the world around her Aranda witnessed the turbulent politics of the Trump era and the repercussions of the January 6 Capitol attack she became motivated to think she should pursue a career in law And it was after a study abroad program, where she studied terrorism and counterterrorism, that she decided for her career path to look instead for artistic ways to serve the world. During her junior year, the Queens native entered Rochester’s Art New York program, interning at a nonprofit gallery in Manhattan’s Chelsea neighborhood called Field Projects Art New York is set up to fully engage students in the workings of the art world in a city rich with galleries and artists they list their top 10 choices of internship sites and start to reach out to those places “The students do a tremendous amount of work up front,” says Heather Layton an associate professor of art who recently served as the program director and its resident faculty member in New York Layton says that fewer than half of the students accepted are studio art majors but they are nonetheless looking to explore opportunities in the art world “It can be difficult to get an internship in New York City,” says Layton Aranda became fascinated with the book art form “Book art can be any kind of documentation or art form encased in a book,” she says “It can be difficult to define an artist’s book because it’s not what you would think of as a novel or a picture book zines have been voices for marginalized communities from the punk zines of the 1970s to the 1990s riot grrrl movement of women’s self-publication Layton says of zines: “They can carry huge ideas in small and portable forms It’s a truly democratic art form in the sense that anyone can make or distribute them Authors bypass publishing companies completely What drew Aranda to zines was how inventive she could be in their design “I like knowing that book art lets you practice any kind of medium while still being consistent in the final product,” says Aranda Aranda saw in book art an opportunity to self-reflect and share the beauty of her childhood community. When students were assigned a mapping project of their first experiences living in New York for the Art New York Field Studio course, she made an artist’s book of her neighborhood in Queens “My neighborhood is where I see myself having grown up Adds Layton: “As you flip through this gorgeous book made on semi-transparent paper and see the overlapping imagery and text Angelica’s hometown neighborhood comes to life Every single one of the things that Angelica does is well done.” During her internship, Aranda also started her first commercial venture. She set up a booth at a festival to support her home community and its businesses, and she sold her first books there. Her next step was to apply for the highly competitive Creatives Build New York (CRNY) program CRNY recognizes the critical role of artists by providing guaranteed income and employment to 2,700 artists living New York State “I was blessed that I got it,” says Aranda who will receive an income of $1,000 for 18 consecutive months with no strings attached “Artists are historians and community builders, and it wouldn’t feel right to take a grant like that and not do something for the community that helped me become who I am,” Aranda says. So she decided to help build up the University’s Sage Art Center community through a project called Mart Crew She began by setting up a drop box at Sage and inviting students and alumni to submit original works of art on a 4-inch x 5-inch piece of paper “She relentlessly recruited for that,” says Layton she scanned the submitted works and presented them in a small artist’s book Included in each book was also an original work of art made by another participant recruited her as a teaching assistant for her performance art course Aranda also taught a zine workshop to her peers hosted in conjunction with the undergraduate Creative Arts Club and the Art and Music Library where she’s currently cataloging and archiving the entire collection of artists’ books under the direction of the art librarian Stephanie Frontz Aranda plans to pursue graduate study in library science “I always want to find a way to give back to the community,” she says I’ll find a way to share what I do with the people I’m around.” Artist Mizin Shin inspires change with printmaking the Rochester professor has harnessed her love of a traditional art form combined with a digital sensibility Funded internships open doors to graduate schools, career paths, and personal growth equitable access to internships helps Rochester students preview their futures Science under the microscope of visual art University of Rochester student Gabrielle Meli brings scientific processes to her art Metrics details Hematopoietic stem cells (HSCs) develop from hemogenic endothelium within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM) To identify the signals responsible for HSC formation here we use single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from hemogenic endothelium to HSCs and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal Pseudotemporal ordering reveals dynamics of gene expression during the hemogenic endothelium to HSC transition identifying surface receptors specifically expressed on developing HSCs Transcriptional profiling of niche endothelial cells identifies corresponding ligands including those signaling to Notch receptors are sufficient to support the generation of engrafting HSCs These studies provide a transcriptional map of the signaling interactions necessary for the development of HSCs and advance the goal of engineering HSCs for therapeutic applications specialized endothelial-like precursor cells simultaneously downregulate endothelial-specific and activate hematopoietic-specific transcriptional programs to give rise to hematopoietic progenitors and HSCs For functional long-term engrafting HSCs to emerge HE must acquire and maintain HSC-defining properties such as the ability to self-renew properties which distinguish rare HSCs from other embryonic hematopoietic progenitors the combination of cell-intrinsic and extrinsic signals necessary and sufficient to support the acquisition of these HSC-defining properties during embryonic development from HE remains inadequately defined knowledge of which is essential for the goal of engineering HSC development ex vivo This argues the need for further efforts to identify the precise signaling interactions between arterialized HE and the arterial vascular niche required for HSC formation This argues the need to study the unique properties of rare HSC precursors using single-cell techniques to study the transcriptional changes associated with HSC formation from HE in the AGM niche at single-cell resolution with a specific focus on genes encoding cell surface receptors and downstream signaling molecules Complementary transcriptomic analysis of the AGM-EC niche identified cognate ligands interacting with receptors on developing HSCs which enabled us to engineer a stromal cell-independent niche that supported the generation of engrafting HSCs from embryonic hemogenic precursors in vitro these studies provide crucial insight into the precise conditions necessary to recapitulate the development of HSCs from HE advancing translational efforts for de novo HSC generation these results demonstrate that the V+61+E+ population while heterogeneous for HSC potential at the single-cell level significantly enriches for rare HSC precursors within the AGM across their developmental spectrum starting from the earliest subset defined phenotypically as HE and a subset of HSC precursors transcriptionally defined as HE (lacking expression of Itga2b/CD41 i Progenitor cell type (expressing Runx1 plus one or more of Flt3 j Gene-set scores for signature genes from (f) in transcriptionally defined cell types (boxplots show median values and interquartile ranges; upper/lower whiskers show 1.5× interquartile range k Gene expression heatmap of Notch receptors and target genes in pseudotime comprising a population enriched for functionally validated HSC precursors successfully captures the developmental emergence of HSCs from HE which is uniquely characterized by overlapping arterial endothelial and HSC-specific transcriptional signatures These results demonstrate the utility of our single-cell transcriptomic analysis to elucidate the developmental dynamics of signaling pathways during the HE to pre-HSC/HSC transition Based on these findings suggesting a role for ligand–receptor integrin interactions via VLA-4 in the AGM-EC niche we also utilized immobilized recombinant fibronectin fragment (FN-CH-296) which specifically binds cell-surface VLA-4 and VLA-5 in vitro this approach successfully identified a group of signaling ligands that when integrated into an in vitro engineered niche was sufficient to support functional HSC generation from embryonic hemogenic precursors Relative skewing of contribution toward early T lymphoid fate and away from B lymphoid and late erythroid fates in engineered conditions suggests relatively increased activation of Notch pathway and/or deficiency of yet undefined signals presented by AGM-EC stroma these results demonstrate that a stroma-independent engineered niche is sufficient to partially recapitulate the vascular niche supporting HSC development from embryonic hemogenic precursors and that further optimization through the incorporation of additional niche-derived signals identified by the ligand–receptor analysis and temporal modulation of key signal pathways such as Notch during culture could further enhance the balance of HSC generation and self-renewal versus lineage differentiation we provide several advances towards deciphering the complex signaling interactions required to support the specification and self-renewal of HSCs during embryonic development using an ex vivo vascular niche modeling the embryonic AGM we identified a combination of phenotypic markers (V+61+E+) that enable enrichment of functionally validated HSC precursors encompassing the transition from HE to pre-HSC/HSCs using scRNA-seq of the primary AGM V+61+E+ population and the progeny of these cells during their maturation to HSCs and self-renewal in the AGM-EC niche combined with computational analysis to analyze arterial EC and pre-HSC/HSC-specific gene expression changes over pseudotime we generated a comprehensive transcriptional map of HSC development from HE we utilized these data to identify receptors and signal pathways expressed during HSC specification and self-renewal and combined with complementary transcriptional analysis of the HSC-supportive AGM-EC niche to identify cognate ligands produced an atlas of potential ligand–receptor interactions regulating HSC development in the vascular niche we applied this knowledge to rationally design a stromal cell-independent engineered niche sufficient to generate engrafting HSC from embryonic hemogenic precursors in vitro Future studies will further determine how differential HSC-supportive properties of AGM-EC may relate to the heterogeneity of the primary niche EC of the aorta from which they are derived including such variables as differential expression of key transcriptions factors and endothelial maturation or activation state Although our studies suggest an important function for CXCL12 in the engineered niched during the initial HE to HSC transition future studies will be required to determine the precise mechanism by which CXCL12 interacts with its receptor(s) to promote stage-specific aspects of HSC generation our studies identified molecular interactions predicted by single-cell transcriptomics to establish unique engineered conditions sufficient to support the generation of engrafting HSCs from embryonic hemogenic precursors Building on this genome-wide map of the “interactome” regulating HSC development in the AGM vascular niche future studies will be necessary to characterize the role of additional niche factors identified by our ligand–receptor analysis in further optimizing HSC generation in vitro particularly from earlier stages of development Based on dynamic changes in the expression of genes involved in key signal pathways we hypothesize that stage-specific modulation of these pathways during culture in the engineered niche will also enable further optimization of HSC specification and self-renewal we hypothesize that signals derived from non-endothelial AGM niche cells such as surrounding mesenchymal populations may also be critical to optimally support HSC generation ex vivo our studies represent a significant step toward precisely defining the niche factors required for HSC generation which will be critical for advancing therapeutic applications such as disease modeling and cellular therapies from human pluripotent stem cells Although the V+61+E+ immunophenotype enabled enrichment of cells with HSC potential at different stages of maturation from HE to HSC in the AGM this population remains functionally heterogeneous at the single-cell level and in silico analysis was required to further resolve cells with HSC potential in our scRNA-seq data although we identified cells co-expressing these markers within intra-aortic hematopoietic clusters of the AGM region by immunostaining this approach is insufficient to localize HSC precursors with any degree of certainty in vivo there are no existing surface markers or single-gene reporters that can be used to confidently localize pre-HSC/HSC at single-cell resolution in the AGM at this stage Future studies that leverage rapidly evolving technologies in spatially resolved single-cell multi-omics will be critical to adding more precise spatial information about how HSCs emerge from HE and the intercellular signaling interactions supporting this process in the AGM in vivo Source and catalog numbers for key reagents and antibodies are listed in Supplementary Table 1 Wild-type C57Bl6/J7 (CD45.2) and congenic C57BL/6.SJL-Ly5.1-Pep3b (CD45.1) mice were bred at the Fred Hutchinson Cancer Research Center C57Bl6/J7 CD45.2 mice were used for timed matings Mice between 6 and 10 weeks of age used for most experiments were housed in individually ventilated and HEPA-filtered microisolator cage environments with room temperature maintained between 21 and 24 degrees Celsius and humidity between 30% and 70% All animal studies were conducted in accordance with the NIH guidelines for the humane treatment of animals and were approved by the Institutional Animal Care and Use Committee at the Fred Hutchinson Cancer Research Center (Protocol #50765) to isolate hemogenic endothelial populations a combination of anti-mouse VE-Cadherin PECy7 with or without additional anti-mouse antibodies for CD45 (PE or FITC) Relevant isotype control antibodies were used to set gates 4′,6-diamidino-2-phenylindole (DAPI) staining was used to gate out dead cells All reagents for cell staining were diluted at 1:200 in PBS with 10% FBS and staining was carried out on the ice or at 4°C Cells were sorted on a BD FACSAria II equipped with BD FACSDiva Software (v9) with index sorting capability sorting was performed in single-cell mode with maximum purity mask settings to minimize contaminating cells and T75 tissue culture plates (pre-treated with 0.1% gelatin) in EC media without added VEGF The first passage to T75 flask was considered passage 0 with subsequent passages every 3–4 days plated at ~5 × 105 cells per T75 flask All AGM-EC derivations were tested by plating cells at 4 × 104 cells/24 wells in X-vivo 20 media to ensure that a confluent layer of viable ECs was maintained for at least 7 days in serum-free conditions Endothelial identity/purity for each independently derived AGM-EC was confirmed by FACS for surface expression of VE-Cadherin independently derived AGM-EC were frozen at low passage at 1 × 106 cells/vial for subsequent experiments For co-culture experiments to compare HSC-supportive capacity AGM-EC from multiple derivations were thawed in parallel from low passage aliquots onto gelatin-treated T75 flasks in EC media above and used at similar passage number for serum-free co-culture assays with freshly sorted hemogenic precursors For single cell-RNA-sequencing (scRNA-seq) experiments AGM-EC from multiple derivations were thawed in parallel from low passage aliquots onto gelatin-treated T75 flasks in EC media Confluent EC was cultured in serum-free medial (X-vivo 20) 24 h prior to harvest by treatment with TrypLE Express washed twice in PBS in 0.04% ultrapure BSA and resuspended PBS with 0.04% ultrapure BSA in on ice for scRNA-seq sorted cells were resuspended in serum-free culture media with cytokines and plated at 1–2 embryo equivalent of cells per 24-well containing AGM-EC hematopoietic progeny were harvested by vigorous pipetting for subsequent analysis by flow cytometry and transplantation assays Multilineage engraftment was defined as >5% donor (CD45.2) contribution to the peripheral blood with the contribution to each lineage of donor myeloid (Gr1 and F4/80) and T cells (CD3) detected at ≥0.5% at 16–24 weeks post transplant there was no stripping step after the final position Slides were removed from the stainer and stained with DAPI for 5 min and coverslipped with Prolong Gold Antifade reagent (Invitrogen/Life Technologies then whole slide images were acquired on an Aperio FL fluorescent slide scanner (Leica Biosystems For single-cell RNA-sequencing (scRNA-seq) studies freshly sorted AGM-derived cells or cells harvested following culture (as indicated above) were washed with PBS containing 0.04% ultrapure BSA and resuspended in 0.04% ultrapure BSA in PBS on ice Cell suspensions were loaded into the Chromium Single Cell B Chip (10X Genomics) and processed in the Chromium single cell controller (10X Genomics) targeting 3500 cells per lane from freshly sorted AGM-derived cells or progeny of AGM-derived cells following culture on AGM-EC or engineered conditions or the remaining 50% progeny of clonal AGM-derived V+E+61+ cells following AGM-EC co-culture and flow cytometry analysis (estimated to contain <500 cells) The 10X Genomics Version 2 single cell 3’ kit was used to prepare single-cell mRNA libraries with the Chromium i7 Multiplex Kit Sequencing was performed for pooled libraries from each sample on an Illumina NextSeq 500 using the 75 cycle targeting a minimum of 50,000 reads per cell scRNA-seq was performed from individual samples from each AGM-EC line resulting in the acquisition of >1000 individual high-quality cell sequences per cell line scRNA-seq was performed in two independent experiments from cells sorted from embryos pooled at E10 (46 embryos resulting in acquisition of >3000 individual high-quality cell sequences between E10 and E11 For clonal progeny of AGM-derived V+61+E+ cells (HSC CFC) generated following AGM-EC co-culture scRNA-seq was performed from two independent resulting in the acquisition of 152 high-quality cell sequences from one colony and 991 high-quality cell sequences from the second colony For the progeny of AGM-derived V+61+E+ cells following culture on AGM-EC or engineered conditions scRNA-seq was performed from cells freshly harvested from each condition processed and sequenced in parallel to minimize batch effects resulting in acquisition of 6887 high-quality cell sequences (from AGM-EC co-culture conditions) and 4871 high-quality cell sequences (from engineered conditions) All sequencing data have been uploaded to NCBI GEO (accession number GSE145886) The Cell Ranger 2.1.1 pipeline (10X Genomics) was used to align reads to the mm10 reference genome and generate a feature barcode matrix filtering low-quality cells using default parameters The Monocle (v.2.99.3) platform was used for downstream analysis (in R v.3.6.1) combining data for cells from each individual sample and/or replicate (as described above) for downstream analysis using a negative binomial model of distribution with fixed variance normalizing expression matrices by size factors Counts for UMI (unique molecular identifiers) and unique genes expressed per cell are shown in boxplots in supplementary figures for each sample (showing median values and interquartile ranges; upper/lower whiskers show 1.5× interquartile range with outliers shown as individual dots) Genes used for clustering were selected based on dispersion thresholds and the preprocessCDS() function was used to project the data onto the top principal components (excluding principal components that contributed little to the overall variance) UMAP was used for dimensionality reduction with the reduceDimension function Clustering was performed by Louvain method with the clusterCells function Violin plots of gene-set scores for each cell type were generated using ggplot function with geom_violin() and geom_boxplot() (boxplots show median values and interquartile ranges; upper/lower whiskers show 1.5X interquartile range) in the ggplot2 package (v 3.3.2) Only cells identified by strict expression of indicated genes are classified as a given cell type by this method This approach was chosen to maximize cell type specificity for downstream analysis of expressed receptors and ligands leaving a subset of cell types unclassified rather than imputing cell types for all cells in the scRNA-seq data set differential gene expression was performed using the differentialGeneTest function with fullModelFormulaStr set to Pseudotime limited to genes with detected expression in >5% of cells and a significance cutoff of q value < 0.01 unpaired Wilcoxon Rank Sum Test (for gene-set scores ggupbr package v0.4.0) was used to calculate P values where indicated results were replicated in at least 3 independent experiments Further information on research design is available in the Nature Research Reporting Summary linked to this article R scripts used for the analysis of scRNA-seq data have been deposited at Github and are publicly available as of the date of publication. Github repository: https://github.com/FredHutch/hadland-etal-2022; Zenodo: https://zenodo.org/record/5806809#.YcphyyyIb0o Many layers of embryonic hematopoiesis: new insights into B-cell ontogeny and the origin of hematopoietic stem cells Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs Definitive hematopoiesis is autonomously initiated by the AGM region Development of hematopoietic stem cell activity in the mouse embryo A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development Notch signal strength controls cell fate in the haemogenic endothelium The expression of Sox17 identifies and regulates haemogenic endothelium NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells Medial HOXA genes demarcate haematopoietic stem cell fate during human development Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium Differentiation of human embryonic stem cells to HOXA Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver Concealed expansion of immature precursors underpins acute burst of adult HSC activity in foetal liver In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus Tracing the origin of the HSC hierarchy reveals an SCF-dependent IL-3-independent CD43(-) embryonic precursor Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos Single-cell transcriptional profiling: a window into embryonic cell-type specification Developmental trajectory of pre-hematopoietic stem cell formation from endothelium Tracing haematopoietic stem cell formation at single-cell resolution Single-cell transcriptomics reveal the dynamic of haematopoietic stem cell production in the aorta Single-cell transcriptomics identifies CD44 as a marker and regulator of endothelial to haematopoietic transition Tracing the first hematopoietic stem cell generation in human embryo by single-cell RNA sequencing Embryonic endothelial evolution towards first hematopoietic stem cells revealed by single-cell transcriptomic and functional analyses Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells A common origin for B-1a and B-2 lymphocytes in clonal pre- hematopoietic stem cells Clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture Iterative single-cell analyses define the transcriptome of the first functional hematopoietic stem cells Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development Sox17 is indispensable for acquisition and maintenance of arterial identity Hedgehog and Bmp polarize hematopoietic stem cell emergence in the zebrafish dorsal aorta Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow Endothelial protein C receptor-expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver Generation and analysis of GATA2(w/eGFP) human ESCs reveal ITGB3/CD61 as a reliable marker for defining hemogenic endothelial cells during hematopoiesis Integrin alphaIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo The Notch target genes Hey1 and Hey2 are required for embryonic vascular development Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods https://www.biorxiv.org/content/10.1101/110668v1 (2017) ERG dependence distinguishes developmental control of hematopoietic stem cell maintenance from hematopoietic specification MLLT3 governs human haematopoietic stem-cell self-renewal and engraftment Kdm6b regulates context-dependent hematopoietic stem cell self-renewal and leukemogenesis Evi1 is essential for hematopoietic stem cell self-renewal and its expression marks hematopoietic cells with long-term multilineage repopulating activity Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence SIRT6 controls hematopoietic stem cell homeostasis through epigenetic regulation of Wnt signaling Single-cell transcriptome atlas of murine endothelial cells p57 is required for quiescence and maintenance of adult hematopoietic stem cells Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence RBPjkappa-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells Gata2 is required for HSC generation and survival Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition Developing HSCs become Notch independent by the end of maturation in the AGM region Fgd5 identifies hematopoietic stem cells in the murine bone marrow Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells A draft network of ligand-receptor-mediated multicellular signalling in human Notch and Wnt signaling in the emergence of hematopoietic stem cells BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development EphrinB2 regulates the emergence of a hemogenic endothelium from the aorta Cannabinoid receptor-2 regulates embryonic hematopoietic stem cell development via prostaglandin E2 and P-selectin activity Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1 Insulin-like growth factor 2 modulates murine hematopoietic stem cell maintenance through upregulation of p57 Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells Multi-layered spatial transcriptomics identify secretory factors promoting human hematopoietic stem cell development Multispecies RNA tomography reveals regulators of hematopoietic stem cell birth in the embryonic aorta Discrete Notch signaling requirements in the specification of hematopoietic stem cells Notch2 governs the rate of generation of mouse long- and short-term repopulating stem cells Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1 Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin Multipotent progenitors and hematopoietic stem cells arise independently from hemogenic endothelium in the mouse embryo Functional identification of the hematopoietic stem cell niche in the ventral domain of the embryonic dorsal aorta Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells Rap1b promotes notch-signal-mediated hematopoietic stem cell development by enhancing integrin-mediated cell adhesion CXCR4 is required for the quiescence of primitive hematopoietic cells Concise review: CXCR4/CXCL12 signaling in immature hematopoiesis-lessons from pharmacological and genetic models Somite-derived retinoic acid regulates zebrafish hematopoietic stem cell formation Activation of the arterial program drives development of definitive hemogenic endothelium with lymphoid potential SDF-1/CXCL12 enhances survival and chemotaxis of murine embryonic stem cells and production of primitive and definitive hematopoietic progenitor cells CXCR4 signaling negatively modulates the bipotential state of hemogenic endothelial cells derived from embryonic stem cells by attenuating the endothelial potential Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4 The chemokine SDF-1 activates the integrins LFA-1 and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells is required for induction of notch signaling circlize Implements and enhances circular visualization in R Wang, Y. et al. iTalk: an R package to characterize and illustrate intercellular communication. bioRxiv https://www.biorxiv.org/content/10.1101/507871v1 (2019) Download references Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under Award Number RC2DK114777 (to I.D.B. And Blood Institute of the NIH under Award Number K08HL140143 (to B.H.) is supported by the American Society of Hematology Scholar Award This research was supported by the Experimental Histopathology and Flow Cytometry Shared Resources of the Fred Hutch/University of Washington Cancer Consortium (P30 CA015704) Flowers for superb technical assistance and members of the Trapnell University of Washington School of Medicine Writing – original draft: B.H.; writing – review & editing: all authors; funding acquisition: B.H. are inventors of a patent related to technology in this manuscript on the use of Notch agonists for expansion of embryonic HSC (US Patent # US10463698B2 Applicant: Fred Hutchinson Cancer Research Center Nature Communications thanks Valerie Kouskoff reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-022-28781-z Sign up for the Nature Briefing newsletter — what matters in science Michael Behn shifted his career focus from aspiring chef to knife sharpener and has never been happier Ask Michael Behn what he loves about knives and you’d better be ready for some serious geeking out on the subject You’ll hear about his passion for Kikuichi knives which draw on Japanese bladesmithing skills dating back 750 years Or he’ll convince you that switching from that dull tool in your kitchen drawer to a high quality knife feels like cutting vegetables with a laser When he started working as a restaurant cook he spent his first $220 paycheck on a quality chef knife “It’s always been about the knives,” says Michael a Filipino American who grew up in the northern suburbs of Atlanta his passion for knives lay dormant as he pursued a career in the restaurant business When asked about his career in the restaurant industry Michael reflects that he only seemed to learn things the hard way he worked his way up through the ranks of the kitchen starting as a dishwasher in a sushi restaurant. He worked at various eateries in Atlanta and around the state exposing him to different cuisines and some big-name chefs he was a sous chef with 35 people working under him He even dreamed of becoming a chef himself But one crucial ingredient was missing from his restaurant life: happiness The razor-thin profit margins forced innovative culinary ideas and high-quality ingredients to be ditched for the generic and cheap It all came crashing down in the COVID-19 pandemic which pushed many restaurants to breaking point With his co-workers talking about jumping ship but things got heated and the chef threw his box of knives outside “I just grabbed my stuff and left,” Michael recalls his love of knives resurfaced in the form of a business idea he had learned how to sharpen knives the traditional Japanese way Now he began offering knife-sharpening services to the Atlanta-area chef community he had grown to know so well but Michael brought something new to the table Whereas some sharpeners used artificially powered belt grinders Michael used a superior method of sharpening which is the most artisanal way to sharpen a knife,” he says he added detailed touches such as wrapping the sharpened knives in Japanese newsprint with anime-branded stickers urging caution with the sharp blades He named the business Moshi Moshi after the Japanese greeting meaning hello that is used when answering the phone The business grew. Initially, Michael drove for hours every day to pick up and deliver knives, but soon customers began dropping them off at his place. He asked a former employer and friend, Chef Kevin Gillespie to feature Moshi Moshi in an Instagram post and immediately got booked for two months straight Through trial and error and a lot of YouTube videos, he got better at his craft. He promoted the business through Instagram posts of him slicing through magazine paper with #supersharp blades He became fluent in sharpening terminology like “burrs” and “grit grades.” “A really well-sharpened knife is just geometry,” he explains “It’s just two flat planes meeting at an acute apex He credits his wife, a CPA and director of tax at a large corporation, with helping the business succeed. Using Intuit QuickBooks, she helps him stay organized and handles the financials and taxes. “She’s pretty much the brains behind it,” he says. “I’m definitely the hands.” Today, Michael teaches others how to sharpen knives and has started franchising Moshi Moshi. Just as vital, he’s found happiness in being kinder to himself and others. Yes, he enjoys working shorter hours and having more financial control over his life than ever before. But a huge driver for him is providing other cooks a way out of unhappy jobs and paying them a living wage. “It matters to pay a fair wage ’cause I never got one,” he says. “The business is not just about making money. We’re doing real stuff here—real stuff being the relationship with the franchisees and interactions with the customers.” View this post on Instagram A post shared by Intuit (@intuit) As a global financial technology platform, Intuit helps self-employed customers like Michael achieve their own dreams Our innovative products and services improve our customers’ lives opening expanded possibilities—whether you’re opening a coffee shop or carving out a niche as the best knife-sharpener in town Celebrate Hispanic Heritage Month by supporting small business owners Mobile home flipping leads to shared prosperity More from Tori Huber Metrics details Stem-cell niches in mammalian tissues are often heterogeneous and compartmentalized; however whether distinct niche locations determine different stem-cell fates remains unclear here we use the mouse hair follicle niche and combine intravital microscopy with genetic lineage tracing to re-visit the same stem-cell lineages we show directly that the position of a stem cell within the hair follicle niche can predict whether it is likely to remain uncommitted generate precursors or commit to a differentiated fate using laser ablation we demonstrate that hair follicle stem cells are dispensable for regeneration which do not normally participate in hair growth re-populate the lost stem-cell compartment and sustain hair regeneration This study provides a general model for niche-induced fate determination in adult tissues The stem-cell niche as an entity of action Stem cells and their niches: integrated units that maintain Drosophila tissues The tortoise and the hair: slow-cycling cells in the stem cell race Coexistence of quiescent and active adult stem cells in mammals Compartmentalized organization: a common and required feature of stem cell niches Hematopoietic stem cell heterogeneity takes center stage Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion The bone marrow vascular niche: home of HSC differentiation and mobilization Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche Detection of functional haematopoietic stem cell niche using real-time imaging Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance Bmi1 is expressed in vivo in intestinal stem cells Interconversion between intestinal stem cell populations in distinct niches Identification of stem cells in small intestine and colon by marker gene Lgr5 Intestinal label-retaining cells are secretory precursors expressing Lgr5 Intestinal stem cell replacement follows a pattern of neutral drift Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells Defining the epithelial stem cell niche in skin A two-step mechanism for stem cell activation during hair regeneration Hair follicle stem cells in the lower bulge form the secondary germ a biochemically distinct but functionally equivalent progenitor cell population Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34 and the existence of two cell populations within an epithelial stem cell niche Long-term renewal of hair follicles from clonogenic multipotent stem cells Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis Capturing and profiling adult hair follicle stem cells Distinct self-renewal and differentiation phases in the niche of infrequently dividing hair follicle stem cells A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages Induction of hair growth by implantation of cultured dermal papilla cells GATA-3: an unexpected regulator of cell lineage determination in skin Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors Redefining the structure of the hair follicle by 3D clonal analysis CK19CreERT knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs A robust and high-throughput Cre reporting and characterization system for the whole mouse brain Identification of the cell lineage at the origin of basal cell carcinoma Dermal papilla cell number specifies hair size shape and cycling and its reduction causes follicular decline Tracing epithelial stem cells during development Mouse differentiating spermatogonia can generate germinal stem cells in vivo An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement Epithelial stem cells and implications for wound repair The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin Molecular dissection of mesenchymal-epithelial interactions in the hair follicle Conditional gene expression in the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA and rTA linked to the keratin 5 promoter Download references Horwich for critical feedback on the manuscript Haberman for technical support with intravital microscopy is a New York Stem Cell Foundation–Druckenmiller Fellow This work was supported by The New York Stem Cell Foundation and by grants to V.G from the American Cancer Society (RGS-12-059-01-DCC) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (1RO1AR063663-01) designed experiments and wrote the manuscript; P.R performed the experiments and analysed the data; K.R.M The authors declare no competing financial interests Scheme of a mouse hair follicle in quiescence Different cell populations reside in defined anatomical compartments In homeostasis the hair follicle undergoes repeated cycles of regeneration Hair growth is fuelled by stem cells in the niche that proliferate and differentiate to form the seven concentric layers of the mature hair shaft and inner root sheath (IRS) whereas a basal epithelial layer called the outer root sheath (ORS) surrounds the entire structure Notice that the seven inner layers expand from the matrix at the interphase with the mesenchymal dermal papilla whereas the ORS has a different mode of growth and expands in the opposite direction A complete hair cycle alternates between phases of rest (telogen) Single hair follicle stem-cell labelling is achieved using a combination of either K19-CreER/Rosa-stop-tdTomato or Lgr5-CreER /Rosa-stop-tdTomato alleles and administration of a single low dose of tamoxifen to achieve a low frequency of Cre-mediated loxP recombination and mosaic expression of the fluorescent tdTomato reporter within the stem-cell niche The lineage of single labelled stem cells is traced in vivo during hair growth Using two-photon laser scanning microscopy we can re-visit the same hair follicles non-invasively in live mice at different stages of hair regeneration Each panel depicts low (top) and high (bottom) magnification images of live hair follicles captured in first telogen Panels depicting the green (left) and red (middle) channel as well as a composite image (right) of a group of follicles in rest phase (telogen) K14-H2BGFP marks all the epithelial cells in the skin including the hair follicles Lef1-RFP marks mesenchymal cells in the dermis including the dermal papilla at the bottom of the hair follicles The tdTomato-Cre reporter (K19- or Lgr5-driven) displays mosaic expression in the stem-cell niche after administering a low dose of tamoxifen Notice that the fluorescent intensity of the tdTomato is several-fold higher and easily distinguishable from RFP in the red channel Individual channels and composite images of a group of follicles three (P23) and five (P25) days after administering a low dose of tamoxifen show a non-leaky expression of the Cre reporter (tdTomato) and a quiescent niche between these time points Single optical sections (top) or 3D volume renderings (bottom) of the outer (ORS; left) or inner (right) hair follicle cell layers as seen in vivo using a K14-driven H2B–GFP fluorescent reporter Single optical sections showing cells marked with the tdTomato-Cre reporter (in addition to K14-driven H2B–GFP) in the outer (ORS; left) and inner (right) hair follicle layers Notice the differences in morphology between cells located in different layers within the hair follicle Examples of in vivo lineage tracing of bulge cells in rest and growth phases of a full hair cycle Arrows point to the location of the original cell and that of its progeny occupying different positions in the niche after a full hair cycle In vivo lineage tracing of a single cell located in the hair germ in rest and growth phases over a full hair cycle In advanced hair growth (anagen) an IRS differentiated lineage can be visualized and it is restricted to one side of the follicle as the original founder cell Examples of in vivo lineage tracing of a single bulge cell in rest and growth phases over two consecutive hair cycles Arrows point to the location of the stem cell that remains uncommitted in the niche during both hair cycles Examples of in vivo lineage tracing of a single bulge cell lineage in rest and growth phases over two consecutive hair cycles A bulge cell positioned in the lower bulge undergoes limited and more extended amplification in the ORS over two consecutive hair cycles After regression of the follicle at the end of the second hair cycle some surviving ORS clones form part of the hair germ before the third hair cycle begins Arrows depict the clonal expansion and contraction of the bulge stem-cell lineage during the two hair cycles a stem cell located in the upper bulge does not commit to a specific fate and is likely to remain quiescent a cell positioned in the lower bulge is more likely to become activated and undergo limited amplification as part of the ORS while still remaining relatively undifferentiated ORS cells that survive the regression phase in one hair cycle can be situated in the niche compartment that becomes the new hair germ Once positioned within the hair germ a cell will commit towards a differentiation pathway generating the cell types necessary to support hair growth Loss of a stem-cell pool due to injury can induce an epithelial cell to enter the niche and contribute to its recovery once a cell enters the niche it is subject to the same inputs as previous resident cells and as a result it will acquire a hair fate and actively contribute to hair regeneration Serial optical sections of a mouse hair follicle captured in vivo by multiphoton microscopy using a K14H2BGFP reporter 3D volume rendering of the tdTomato Cre reporter showing the spatial distribution of ORS clones during hair growth Time-lapse recording of a hair follicle in growth (Anagen IIIa) as seen using a K14H2BGFP reporter Notice the spatially restricted mode of proliferation and migration Time-lapse recording of a hair follicle in growth (Anagen IV) as seen using a K14H2BGFP reporter Time-lapse recording of a hair follicle in growth (Anagen IIIa) using a K14H2BGFP reporter showing an example of ORS tdTomato+ clones being separated due to active cell migration Time-lapse recording of a regions above the hair follicles that previously had their bulges ablated Time-lapse recording of a live hair follicle 24 hours after bulge ablation as seen using the K14H2BGFP and Lef1RFP reporters Serial optical sections of mouse hair follicles captured by multiphoton microscopy in vivo using K14CreER/tdTomato Notice the biased expression of the Cre reporter toward epithelial layers above the hair follicle niche Download citation Nature Reviews Molecular Cell Biology (2024) By combining live imaging and single stem cell/progeny labelling in live mice Valentina Greco and colleagues show that cells located in different compartments of the hair follicle stem-cell niche have different cell fates Laser ablation shows that multipotent bulge stem cells are not essential for hair regeneration because epithelial cells can re-populate the lost stem-cell compartment These findings demonstrate that the position of a stem cell within its extended niche controls its long-term fate and behaviour This may be a general mechanism for the regulation of other adult stem cells Many advisors start their careers taking on anyone and everyone as clients the long-term effects of this choice can hamper a practice’s growth for years to come Twenty small or nonideal clients might be manageable for an advisor or team but 100 of these clients could hinder a firm We coach advisors to think through their ideal clients from the very beginning, so they can build their expertise around a group they enjoy and achieve success in this niche. More specifically, we work with them to develop a niche, a focused clientele group that the advisor is uniquely able to serve Not only does specializing in a niche offer greater opportunities for growth but it means you have a proactive strategy for capturing business from a market segment you want to work with Considerable research shows that having a clear focus is great for business. Studies from Kitces Research in 2020 found that top advisors with a niche (versus top advisors without a niche) serve an average of 14% more clients and have clients with an average of 25% more investible assets and higher net worth A practice with more clients (who have more assets and higher net worths) certainly sounds like something worth working toward time-strapped advisors are always looking to do more with less you can gain expertise and experience to scale more effectively Kitces Research also revealed that advisors with niches spend 150-plus more hours every year on high-value client-facing activities—or 28% more time with clients and prospects — while spending 13% less time doing back-office tasks This additional time spent with clients and prospects can lead to deeper relationships Specializing in a niche empowers you to be strategic about the clients you want to serve and it’s especially useful if you adopt a virtual mindset or want to take on virtual clients If you have expertise in something your niche needs people can then find you and work with you from anywhere remember to keep these three things in mind: Advisors need to be able to locate the people in their niche We know advisors who have niches in municipalities We also know advisors who focus on specializations like funeral homes and car dealerships which are bit more esoteric (but can be highly profitable) It’s important to focus on a niche that will enable you to use your services and pay your fees there are many ways to help people who aren’t ideal clients through financial literacy programs an advisor who worked at an ice cream stand during the summers and as a waitress during the school year became the go-to person for seasonal and cash businesses in her area Niche advisors are specialists who have expertise in solving particular problems for a very select group of people with similar needs so it’s important that you carefully sift and sort out what that focus should be for your practice If you’ve thought about exploring a niche in the past and you want to set up your business for success start creating a plan that will get you there in the New Year [More: The sky’s the limit for an adviser dedicated to working with airli ne pilots] Kristine McManus serves as chief advisor growth officer at Commonwealth Financial Network. Metrics details Granulocyte recruitment to the pulmonary compartment is a hallmark of progressive tuberculosis (TB) This process is well-documented to promote immunopathology but can also enhance the replication of the pathogen Both the specific granulocytes responsible for increasing mycobacterial burden and the underlying mechanisms remain obscure We report that the known immunomodulatory effects of these cells such as suppression of protective T-cell responses play a limited role in altering host control of mycobacterial replication in susceptible mice we find that the adaptive immune response preferentially restricts the burden of bacteria within monocytes and macrophages compared to granulocytes mycobacteria within inflammatory lesions are preferentially found within long-lived granulocytes that express intermediate levels of the Ly6G marker and low levels of antimicrobial genes These cells progressively accumulate in the lung and correlate with bacterial load and disease severity and the ablation of Ly6G-expressing cells lowers mycobacterial burden These observations suggest a model in which dysregulated granulocytic influx promotes disease by creating a permissive intracellular niche for mycobacterial growth and persistence we used multiple mouse models of susceptibility to investigate how neutrophil infiltration promotes Mtb growth discovering a subset of lung granulocytic cells that are permissive to Mtb infection a Mouse models of increasingly severe disease (left to right) with representative scatterplots of CD11bHigh Ly6GPos pulmonary cells at 28 days PI and quantified (far right) as percent Ly6Gpos cells by recovered CFUs in the lung of each individual mouse b Representative scatterplots (left) and quantification (right) of CD11bHigh Ly6GHigh neutrophils (top right) CD11bHigh Ly6GMid granulocytes (middle right) and CD11bHigh Ly6GNeg monocytes/macrophages (bottom right) in Bl6 WT or Nos2-/- mice at the indicated time points PI N = 4 mice per group and error bars represent standard deviation between biological replicates within each group c Multiplex analysis of the indicated cytokines and chemokines in the lungs of Bl6 WT or Nos2−/− mice at the indicated time points PI Heatmap represents the average pg/mL of detected cytokine/chemokine from 4 mice per group scaled by row a CFUs per lung at 25 days PI within Bl6 WT or Nos2−/− mice treated with anti-Ly6g depleting antibody (1A8) or isotype control (2A3) b Representative scatterplots of lung cells from infected Bl6 WT (top mid) or Genista (top right) mice treated with aminoguanidine (AG) or Ly6GHigh cells (bottom right) per lung at 56 days PI with or without AG treatment c Quantification of the indicated immune cells in the lungs of AG-treated Bl6 WT mice or AG-treated Genista mice at 56 days PI N = 3–7 mice per group and error bars represent standard deviation between biological replicates within each group Statistical values based on unpaired Student’s T-test or one-way ANOVA with Tukey’s Multiple Comparison analysis we conclude that the disease-attenuating phenotype observed upon Ly6GPos cell depletion is directly attributable to loss of Ly6GPos granulocytic cells a Number of Ly6GPos cells (top) or CFUs (bottom) in the lungs of the indicated mouse strains at 28 days PI b Schematic diagram of mouse chimerism to produce 50:50 WT:Cxcr2−/− mice (where the Ly6GPos compartment is IL10 sufficient) or 50:50 Il10−/−:Cxcr2−/− mice (where the Ly6GPos compartment is IL10 deficient) c Representative plots of CD11bHigh cells from peripheral blood verifying ~50:50 chimerism of WT (CD45.1Pos):Cxcr2−/− (CD45.2Pos) or Il10−/− (CXCR2Pos):Cxcr2−/− (CXCR2Neg) d CFUs recovered from the lungs or spleen of AG-treated chimeric or control mice at 28 days PI e Representative plots of CD11bHigh Ly6GPos cells recovered from infected Nos2−/− mice verifying the isolation of subpopulations via GR1 expression f Total IFNγ production by activated T-cells co-cultured at a 1:1 ratio over 24 h with Ly6GHigh or Ly6GMid cells of the indicated genotype recovered at 28 days PI N = 4 mice per group and error bars represent standard deviation between these biological replicates Statistical values based on one-way ANOVA with Tukey’s Multiple Comparison analysis even when granulocyte recruitment was augmented by AG treatment these data indicated that Ly6GPos cells may contribute to total IL10 levels in the lungs but their IL10 production does not play a significant role in controlling antimicrobial immunity in this model while functionally different subsets of granulocytes may infiltrate the lungs based on the root cause of susceptibility MDSC-based T-cell suppression does not explain the disease-promoting effect of Ly6GPos cells in C3HebFeJ and Nos2−/− mice we quantified the number of bacteria in various niches including myeloid cell subsets and extracellular sites representing significant replicative sites in necrotic lesions a Ratio of extracellular/intracellular CFUs recovered from the lungs of msfYFP-Mtb infected Bl6 WT or Nos2−/− mice at the indicated time points Error bars represent standard deviation between mice within each group left Y-axis) or normalized YFP Units (triangles right Y-axis) in the lungs of msfYFP-Mtb infected Bl6 WT (top) or Nos2−/− (bottom) mice at the indicated time points c YFP units (Y-Axis) as a function of CFUs (X-axis) and Ly6GPos cell number (width of circles with numerical label) in the lungs of infected Bl6 WT or Nos2−/− mice “R2” indicates the correlation coefficient for the fitted line d Number of YFP Units per cell in msfYFP-Mtb associated CD11bHigh Ly6GHigh or CD11bHigh Ly6GNeg cells from Bl6 WT (blue bars) or Nos2−/− (pink bars) mice at the indicated timepoints Inset: YFP Units per cell within Ly6GHigh or Ly6GMid compartments at 35 days PI graphed on a log Y-axis for ease of visualization These data are consistent with a model in which the protective adaptive response preferentially restricts bacterial growth in Ly6GNeg monocytes/macrophages verses Ly6GPos granulocytic cells and recruitment of these more permissive granulocytes promotes infection a Schematic of in vivo pulmonary cell half-life assay (top) and representative histogram overlays (bottom) of Bl6 WT pulmonary CD11bHigh Ly6GPos cells or CD11bHigh Ly6GNeg monocytes/macs left untreated (red) or stained with CellTraceTM Violet via tracheal aspiration and measured at 1 h (gold) or 48 h (blue) post-labeling b Mean half-life of total or c infected YFPPos (red) vs uninfected YFPNeg (black) Ly6GNeg monocytes/macrophages or Ly6GHigh neutrophils in the lungs of Bl6 WT or Nos2−/− mice Error bars represent standard deviation between half-life values calculated from at least 3 separate pairs of biological replicates (mice) N = 3 mice per genotype per time-point post-treatment Statistical values based on two-way ANOVA with Tukey’s Multiple Comparison analysis a Bi-clustering heatmap visualizing the expression profile of the top 30 most differentially expressed genes as sorted by adjusted p-value via plotting the log2 transformed expression values in each sample b Pathway analysis of differentially expressed genes predicting activation or inhibition of upstream regulators in relation to functional neutrophil migration (top) or degranulation (bottom) Ly6GHigh peripheral blood neutrophils were recovered from uninfected Bl6 WT mice (N = 2) Ly6GMid granulocytes were recovered from Bl6 WT mouse lungs at 32 days post infection (N = 3) as sorted by adjusted p-value via plotting the log2 transformed expression values in each sample between of Nos2−/− Ly6GMid vs to Nos2−/− Ly6GHigh lung cells b Differentially expressed genes were significantly enriched in the depicted inflammatory signaling pathway Cells were collected from mouse lungs at 32 days PI we investigated the phenotype and role of these cells in Mtb-susceptible mouse strains Our data indicate that Ly6GPos cells directly promote bacterial replication and can serve as a primary niche for Mtb in susceptible hosts support a model in which the recruitment of granulocytes provides a favorable site for Mtb replication These data indicate that Ly6GPos cells directly promote Mtb growth this effect was not generalizable to Bl6 WT This difference was particularly surprising for 129SvPas and C3HeBFeJ mice as susceptibility in both settings is due to a type I IFN response We conclude that suppressive functionality depends on genetic background but is not required for granulocytes to promote mycobacterial replication While heterogeneity complicates precise characterization of granulocyte subsets our data indicate that Ly6GMid cells are abundant in susceptible mice possess a lifespan at least as long as macrophages poorly express MRTF-SRF-dependent antimicrobial genes and become a major bacterial reservoir even in the context of an immune response capable of controlling Mtb in macrophages interrupting this process may represent a more promising interventional strategy than those targeting upstream mediators and were sex and age matched within individual experiments Ly6GHigh or Ly6GMid cells were isolated from the lungs of WT C57BL6 or 129SvPas mice at 28 days PI using the Miltenyi MDSC Isolation Kit P25 CD4Pos T-cells were pre-activated for 6 h in complete media containing interleukin-2 and CD3/CD28 DynabeadsTM (ThermoFisher Scientific then incubated alone or in 1:1 co-culture with isolated granulocytes in 96-well plates (105 total cells/well) at 37 °C and Ab-stained to verify dye restriction to the pulmonary compartment Half-life values for individual cell populations were calculated by quantifying the number of dyePos cells within mice of each cohort (relative to untreated controls) using a flow cytometer then calculating the linear regression of dyePos cell ablation over time using the formula: ((Ln(2)*Z hours post-treatment)/(Ln(mean # of dyePos cells at 1 h post-treatment/mean # of dyePos cells at Z hours post-treatment))) The calculated cell half-lives were averaged within each genotype and cohort (biological replicates for each time point = 3 mice per cohort per genotype technical replicates for each time point = 9) Any mouse with < 10% of total cells dyePos was discarded No dye signal was observed at 72 h post-treatment For each YFPPos population within each mouse the lowest recorded YFP fluorescence value above background was set equal to 1 YFP Unit All subsequent YFPPos events within the population were divided by this lowest positive value to generate bins Histograms were generated with FlowPy graphing software plotting the number of YFPPos cells falling within each respective bin Approximately 1.25 million Ly6GHigh or Ly6GMid cells were isolated from naïve mouse blood or Mtb infected mouse lungs using the Miltenyi MDSC Isolation Kit RNA from cells in each test group was extracted First and second strand cDNA was synthesized and end-repaired by 5′ phosphorylation and 3′ dA-tailing Adapters were ligated onto the cDNA fragments which were then PCR enriched and sequenced via Illumina (San Diego Resulting reads were trimmed to remove possible adaptor sequence contamination and nucleotides with poor quality were discarded using Trimmomatic v.036 Trimmed reads were mapped to the Mus musculus GRCm28 reference genome using STAR aligner v.205.2b Unique gene hit counts were calculated using featureCounts from the Subread package v.1.5.2 counting only those unique reads that fell within exon regions Gene hit counts between test groups were compared using DESeq2 The Wald test generated p-values and log2 fold changes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 defining differentially expressed genes between comparisons Predictive pathway analysis was performed using Quigen (Hilden Germany) Ingenuity Pathway Analysis software which compared significantly different –log2 gene expression values between test groups Murine cytokine concentrations in filter sterilized lung tissue lysate were 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Charlotte Reames and Kadamba Papavinasasundaram for technical assistance; and the biosafety level 3 and the flow cytometry core facilities at UMMS This work was supported by the National Institutes of Health (grants to C.M.S Department of Microbiology and Physiological Systems University of Massachusetts Medical School Department of Immunology and Microbial Disease Pre-publication disclosure of data: Portions of this manuscript were publically presented at the February 2015 Keystone Symposia on Tuberculosis in Santa Fe at the July 2016 Gordon Research Conference on Microbial Toxins and Pathogenicity in Waterville Valley and at the January 2017 Keystone Symposia on Tuberculosis in Vancouver Download citation DOI: https://doi.org/10.1038/s41385-020-0300-z Sorry, a shareable link is not currently available for this article. Volume 2 - 2022 | https://doi.org/10.3389/fopht.2022.869046 This article is part of the Research TopicInsights in Inflammatory Eye Diseases: 2021View all 7 articles The microenvironment of the CNS (eye and brain) is fertile ground for infection if the barriers are breached The result of pathogen invasion is often devastating destruction of tissues inflammation is broadly classified either as “infectious” (i.e caused by infection) or “non-infectious” which clinically appear to be “non-infectious” turn out to be initiated by infectious agents suggesting that pathogens have been retained in latent or persistent form within ocular tissues and have reactivated to cause overt disease A similar pathogenesis applies to latent infections in the brain Not all CNS tissues provide an equally protective niche while different pathogens escape detection using different strategies This review summarises how immune privilege (IP) in the CNS may be permissive for latent infection and allow the eye and the brain to act as a reservoir of pathogens which often remain undetected for the lifetime of the host but in states of immune deficiency may be activated to cause sight- and life-threatening inflammation The host-pathogen interaction is not an easy one Either the host or the pathogen fails to survive A good outcome for the host is one in which the pathogen is completely cleared Whether this is achieved depends on the fitness of the host and the potential for the pathogen to wreak damage (virulence) Less virulent pathogens have evolved strategies to evade the host’s immune defences and these are variously described as latency This ensures species survival for the pathogen assisted by a conducive host environment This review considers the possibility that the IP status of the CNS offers pathogens special license to evade the immune system The concept of IP developed as an explanation for a phenomenon that did not fit with the emerging dogma on immunological tolerance (IT). IT, as a theory, developed from studies of allograft rejection and the discovery of MHC antigens, and was formulated on the notion of self-non-self-discrimination (SNSD) (6) Immunity to foreign antigens was viewed predominantly in terms of the specificity of adaptive immune responses and SNSD-based IT was the process whereby the immune system avoided T and B cells turning on the host can thus potentially activate the immune system but only do so in the right context (co-stimulation) and microenvironment apparent non-infectious causes of intraocular inflammation are just about as frequent as infectious causes non-infectious IOI is the label for conditions in which no infectious agent can be detected Microbial latency is described operationally as a state “in which host damage that occurs does not perturb homeostasis to a degree that results in clinical disease” (45) An important difference between colonisation and latency is time: colonising microbes are eventually cleared or cause overt disease while latent microbes usually persist for the lifetime of the host frequently without causing disease as for instance with Epstein Barr virus (EBV) Latent infections can be caused by a wide range of microorganisms and occur in many sites. They have a predilection for residency in the CNS, either in parenchymal tissues per se or in the brain and eye border regions (meninges and uveal tract) (10) The following sections illustrate various forms of microbial latency in the CNS and eye Toxoplasma gondii (Tg) is the most common parasitic infection of the eye and in some regions such as Brazil is the commonest cause of IOI (59). Infection is acquired at any age mostly through ingestion of infected meat. Serological evidence of exposure to Tg increases with age and in some countries can reach levels of 80-90% of the population. However, most individuals are asymptomatic, or experience only a mild GI disturbance (60, 61) the complexity of the immunological machinery required to keep Tg bradyzoites in latency is critically dependent on the CNS microenvironment Numerous other parasites, nematodes, cestodes and trematodes, as well as helminths are known to cause ocular infections [reviewed in ref (73)], but most would not be classified as latent infections. However, they often induce minimal inflammation as in the case of intraocular loiasis (74) or in cases of DUSN (75) prior to onset of symptoms Loa loa) might be regarded as having a symbiotic mutualistic or even a commensal relationship with the intraocular compartment such parasites would likely induce a vigorous immune response and be rapidly cleared by the immune system Reactivation of EBV in situ would thus cause extensive damage at the cortical level Interestingly, “subclinical” virus reactivation in the form of viral shedding (for instance in the urine) is a common phenomenon and is exacerbated by many forms of stress (107) Ocular TB is a classic example of latent infection safely sequestered in macrophages: latent TB IOI disease in its miliary form does not contain replicating bacilli while the choroidal tuberculoma may contain replicating microbes and be “infectious” in the true sense Figure 1 Choroidal tuberculoma* in HIV +ve patient two years after cessation of anti-retroviral therapy retroperitoneal) and spleen; regression of initial lesion (white arrow) but appearance of a new lesion (dashed arrow) after six months of anti-TB and anti-retroviral therapy (B); anti-TB therapy stopped at 12 months; complete resolution of both lesions noted at 18 months (C) Disease reactivation involving the CNS parenchyma occurs particularly in states of relative immunosuppression but is more commonly restricted to the CNS border regions Despite large numbers of latently infected people <10% develop overt disease: this suggests that persistence of latent Mtb is the norm in immunocompetent individuals and that the site of latency is frequently the CNS border regions (meninges and uveal tract) causes most if not all forms of IOI and that its aetiology is only masked by the limitation of the diagnostic toolkit warrants consideration The following paragraphs address some clinical IOI entities Choroiditis as a discrete entity is uncommon and takes several clinical forms including multifocal choroiditis and serpiginous choroiditis. Tuberculosis is strongly implicated in serpiginous disease (144) while several infectious aetiologies have been linked to cases of acute posterior multifocal placoid pigment epitheliopathy (APMPPE) (145–153) Although in developed countries, a microbial aetiology for non-infectious uveitis is often elusive, the door to an infectious aetiology has been left open by the re-branding of “non-infectious” IOI to “undifferentiated” IOI (38) this terminology admits that cases of IOI/uveitis in which a microbial aetiology cannot be identified also cannot be differentiated clinically from cases of infectious IOI and despite the range of clinical presentations the commonality between infectious and “non-infectious” is striking infectious agents initiate disease by entry across mucosal or skin barriers in which the primary infection may or may not be symptomatic If the initial infection is not completely cleared it is likely that one of the five potential outcomes of this initial interaction occurs: infection Both disease and latency outcomes require systemic microbial dissemination and localisation to the target site For many of the clinical conditions described above reactivation from latent infection is the most likely pathogenetic explanation since latency is a function of both the CNS microenvironment and a robust immune response It is this immune response which promotes latency via both CD4+ and CD8+ T cell activity for the lifetime of the host Only when immunity declines (immunosuppression aging) does the latent microbe reactivate and replicate causing We argue therefore that most if not all cases of IOI are caused by a host-microbe interaction and the nature of tissue damage varies dependent upon whether the microbe is replicating and causing direct tissue necrosis (e.g CMV retinitis) or whether there is an exaggerated immune reaction to the pathogen as occurs in reactivated Ebola and Mtb latent uveitis Figure 2 Host microbe interaction: the precarious relationship between immune privilege (IP) and latent infection Microbial challenge and invasion of the host through external barriers (cartoon on right) causes acute inflammation and spread of infection with one of three outcomes (box in centre) The host survives if the infection is fully cleared or if the infection becomes latent Keeping microbes in a latent state depends not only on a conducive (“privileged”) microenvironment such as that in the CNS but on a sustained immune response The microbe can reactivate when IP is breached by immune dysregulation (cartoon on left) When the system is perturbed as occurs on pathogen challenge, the organism acts to clear the pathogen and restore homeostasis, achieved through inflammation (17) This may be completely silent (asymptomatic infection) or life-threatening its clearance of the pathogen is unlikely to be complete and persistent (latent) pathogens in the host continue to drive the homeostatic (immunogenic) response this may take the form of overt inflammation (IOI) or may be silent and the eye is an especially sensitive sensor of this response as for instance with the “pepper-and-salt” pigmentary retinopathy of congenital rubella or childhood giardiasis It is no surprise therefore that “undifferentiated” IOI is similar to infectious IOI and continue to harbour them within our cells and tissues in apparent good health until All authors critically revised and edited the manuscript and provided expert input All authors contributed to the article and approved the submitted version University of Aberdeen Development Trust/Saving Sight in Grampian: grant number RG16220-10 The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: John V. Forrester, ai5mb3JyZXN0ZXJAYWJkbi5hYy51aw== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details Bone tissue undergoes constant turnover supported by stem cells Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones Craniofacial bones are flat bones derived from a different embryonic origin than the long bones The identity and regulating niche for craniofacial-bone MSCs remain unknown we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones give rise to all craniofacial bones in the adult and are activated during injury repair Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth indicating that these cells are an indispensable stem cell population Twist1+/− mice with craniosynostosis show reduced Gli1+ MSCs in sutures suggesting that craniosynostosis may result from diminished suture stem cells Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair Jr Recent advances in craniofacial morphogenesis Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow Mesenchymal and haematopoietic stem cells form a unique bone marrow niche Gremlin 1 identifies a skeletal stem cell with bone Stromal cells and stem cells in clinical bone regeneration Advances of mesenchymal stem cells derived from bone marrow and dental tissue in craniofacial tissue engineering Cell sources for bone regeneration: the good Periosteum: biology and applications in craniofacial bone regeneration Cell replication in craniofacial periosteum: appositional vs Bone repair cells for craniofacial regeneration Craniosynostosis: imaging review and primer on computed tomography Craniosynostosis: molecular pathways and future pharmacologic therapy Cranial suture biology: from pathways to patient care Genetic basis of single-suture synostoses: genes Genetics of craniosynostosis: review of the literature Advances in surgical approaches to the upper facial skeleton Craniofacial dysostosis syndromes: evaluation and staged reconstructive approach Surgical treatment of single-suture craniosynostosis: an argument for quantitative methods to evaluate cosmetic outcomes Current treatment of craniosynostosis and future therapeutic directions Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor Absence of endochondral ossification and craniosynostosis in posterior frontal cranial sutures of Axin2−/− mice Bregmatic wormian bone and metopic synostosis Ex vivo model of cranial suture morphogenesis and fate Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling Ihh and Runx2/Runx3 signaling interact to coordinate early chondrogenesis: a mouse model Hedgehog signaling antagonist GDC-0449 (Vismodegib) inhibits pancreatic cancer stem cell characteristics: molecular mechanisms a basic helix-loop-helix transcription factor Craniosynostosis of coronal suture in twist1 mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture Genetic causes of syndromic craniosynostoses Genetic heterogeneity of Saethre–Chotzen syndrome Mutations of the TWIST gene in the Saethre–Chotzen syndrome Integration of FGF and TWIST in calvarial bone and suture development Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche Tissue engineering solutions for tendon repair Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor A review of hedgehog signaling in cranial bone development Twist haploinsufficiency in Saethre–Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFα expression and caspase-2 activation Pivotal role of Twist in skeletal biology and pathology Stem cells and bone: A historical perspective Concise review: hitting the right spot with mesenchymal stromal cells Tissue interactions with underlying dura mater inhibit osseous obliteration of developing cranial sutures The role of TGF-β signaling in regulating chondrogenesis and osteogenesis during mandibular development and fgf-2 modulation of posterofrontal/sagittal suture-derived mesenchymal cells in vitro Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities is required for initial Shh signaling and ectopic activation of the Shh pathway Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning Homeostasis and effector function of lymphopenia-induced ”memory-like” T cells in constitutively T cell-depleted mice Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation Generalized lacZ expression with the ROSA26 Cre reporter strain Generating green fluorescent mice by germline transmission of green fluorescent ES cells Download references Samuels for critical reading of the manuscript and M Shi for technical support on the FACS analysis acknowledges training grant support from the National Institute of Dental and Craniofacial Research This study was supported by grants from the National Institute of Dental and Craniofacial Research Hu Zhao, Jifan Feng, Thach-Vu Ho, Weston Grimes, Mark Urata & Yang Chai carried out most of the experiments and analysed the data participated in the suture cell culture experiments (a–c) MicroCT images of one-month-old wild type mice Each craniofacial bone is labelled with a different color (d–f) HE staining of the sagittal suture (d) (g) Schematic drawing of suture organization (a–m) LacZ staining of craniofacial sutures of one-month-old Gli1-LacZ mice Gli1+ cells are detectable in the mid-suture mesenchyme of the lambdoid (a) basosphenoid-squamous (l) and basosphenoid-frontal (m) sutures (n–o) Immunohistochemical staining of osteogenic differentiation markers Sp7 or Runx2 and lacZ staining (βGal) of craniofacial sutures of one-month-old Gli1-LacZ mice Arrows indicate positive Sp7 or Runx2 signal (p) Whole mount LacZ staining of the posterior frontal suture of 8-day-old (P8) Gli1-LacZ pups p-b′) are sections of the posterior frontal and sagittal sutures and their positions are shown with the arrow and arrowhead (q) LacZ staining of the posterior frontal suture of one-month-old Gli1-LacZ mice Fluorescence imaging of sutures in Gli1-CreERT2;R26tdTomatofl mice one week (a–n) and one month (a′-o′) after induction at 1 month of age Sutures visualized include the lambdoid (a,a′) basosphenoid-squamous (l,l′) and basosphenoid-frontal (m,m′) (n,n′,o′) Immunostaining of Sp7 or Runx2 in the osteogenic front of Gli1-CreERT2;R26tdTomatofl mice Arrowheads indicate Sp7+ or Runx2+ cells in the osteogenic front (a) LacZ staining of the parietal bone of one-month-old Gli1-LacZ mice (b) Percentage of suture Gli1+ cells in the parietal basosphenoid and squamous bones of one-month-old Gli1-LacZ mice (c–d) Visualization of Gli1+ cells in Gli1-CreERT2;R26tdTomatofl mice induced at 1 month of age Gli1+ cells are detectable in the marrow space of the basosphenoid bone (arrows in c) osteocytes close to the bone marrow space are also labelled (arrows in d) although blood cells in the bone marrow are not (a–h) MicroCT images of incisors of Smoothenedflox/flox (control) and Gli1-CreERT2;Smoothenedflox/flox (Smo ICKO) mice induced at one month of age and analysed two months later Arrows indicate normal calcified tissue and arrowheads indicate disrupted calcified tissue in sagittal (b,f) and cross (c,d,g,h) sections (i–l) HE staining of incisors in control (i,k) and Smo ICKO (j,l) mice Normal and disrupted enamel and dentin formation are indicated by the arrow and arrowhead Asterisks indicate periodontal tissue defects (m–o) EdU incorporation and TUNEL assays in the incisors and sagittal sutures of control and Smo ICKO mice induced at one month of age and analysed one month later (n) Quantification of the relative numbers of EdU+ cells (p) Lineage tracing analysis in the sagittal suture parietal bone and palatal suture of Gli1-CreERT2;Smofl/fl;R26ZsGreenfl mice induced at one month of age and analysed two months later (q–x) LacZ and Alizarin Red staining of MSCs from the suture mesenchyme of one-month-old Gli1-LacZ mice either untreated (ctrl) or treated with IHH or GDC0449 (t) Quantitation of Edu incorporation and TUNEL assays of the same MSC cultures (x) Real-time PCR of osteogenic differentiation markers including ALPase Sp7 and Osteocalcin of the same MSC cultures ANOVA was performed and P values were indicated in the figure (y–z) Gli1-CreERT2;R26DTAfl/fl;R26ZsGreenfl and control (Ctrl) mice were induced at one month of age and analysed two months later Fluorescently labelled cells in the fused sagittal and coronal sutures indicate cell ablation is not 100% efficient Dotted lines outline the dental epithelium or bone (a) Gli1+ MSCs within the suture mesenchyme give rise to the osteogenic front These MSCs also give rise to the osteocytes either directly in the osteogenic front region or indirectly through the periosteum or dura (b) IHH secreted from the osteogenic front regulates the differentiation of Gli1+ MSCs in the suture mesenchyme Reprints and permissions Download citation Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. cat food no longer a niche marketGrain-free dry dog and cat food formulas continue to gain market share Learn what these formulas contain and what brands are investing in this movement a database of the ingredient decks of dog and cat foods on sale in the United States we did a thorough analysis of the recipes to determine just how widespread the grain-free movement is in pet food The percentage of recipes using these common alternatives to grains is nearly the same between dry dog and dry cat foods the numbers are similar — 47 percent of the recipes do not contain grains 22 percent have grain-free in their product or formula name 19 percent contain chickpeas and 14 percent contain lentils Of the brands offering dry cat food in the United States 73 percent have at least one product that with a grain-free recipe This analysis of the number of grain-free recipes is only one of many that can be performed using the Dog and Cat Food Ingredient Center. The center contains the collected ingredient decks of dry and wet dog and cat food marketed in the United States in a searchable database ingredient and combination of included or excluded ingredients See live demonstration of database in upcoming webinar What: Speeding product research with the Dog and Cat Food Ingredient Center webinar Register: https://goo.gl/cBhnKS By submitting this form, you acknowledge that use of your data is governed by our Privacy Policy. you agree to receive texts or calls regarding your subscription or other WATT products and services Please call +1 (847) 400-5960 for custom support ‘Black Feminist Guide to the Human Body,’ the playwright and professor seeks a softer life for Black women as they age running at Austin’s Vortex Theatre April 12-May 4 In a society with taboos about discussing the realities of aging Thompson’s production is about taking good care of the changing body Black studies professor Beatrice “Bea” Free confronts her past and the parts of herself she wants to ignore as she drafts a paper titled “The Black Feminist Guide to the Human Body.” With guidance from her body (Cee Cee) and soul (Dee) Bea winds up writing a love letter to Black women and girls out of her memories The result is equal parts spiritual mixtape she was inspired to create the production after seeing the litany of women president of Volunteer State Community College Neither had made it to 75 years old. I’m realizing that here are women who did all the right things and still ended up prematurely passing away,” Thompson said “I wanted to pay homage to the people who put me in a very beautiful place as a scholar and an artist to let people younger than me know that it happens very fast—first you’re the cool young professor Passionately advocating for healthy aging while confronting the alarming health disparities for Black women The Black Feminist Guide to the Human Body encourages Black women to find joy and wisdom in aging rather than despair and to support each other to intentionally aspire to the “softer life.” As Thompson put it “We need to be resting and napping and not always grinding out here And now I understand rituals of care in a different way This choreopoem of original music, dance, and storytelling was commissioned by the National Performance Network, The Vortex, Fusebox Festival and Pyramid Theatre. Each performance is an interactive theatrical experience with audiences invited to use the show’s hashtag and post notes with advice and prayers with the characters to create different versions of the production and medical experts) will participate in talkbacks and health fairs and other wellness events will complement the show’s run from April 12 to May 4 (The first weekend is part of the Fusebox Festival of experimental new work in Austin.) This kind of work has been the focus of Vortex co-founder and producing artistic director Bonnie Cullum since the theatre opened in Austin’s historically Black Six Square in 1988 unashamed art to create action in a shifting age and elevate inclusive discourse from our cultural harbor in Austin alternative theatre company is not something you find every day but over the years Cullum has found that risk has brought reward when we tried things artistically that were a little outside the box what’s different—when we did those things because people came to see things that were more experimental.” Thompson has had two other shows premiere at The Vortex: Underground in 2017 a political thriller that debates the best road to Black liberation; and The Mamalogues in 2019 a satirical comedy about the struggles of Black middle-class single moms to raise healthy Black children in a racist Cullum said she sees Thompson finding her voice as an artist in a way she envisioned when she first started The Vortex “Lisa is creating a piece that is not only urgent but very vulnerable and self-revelatory,” Cullum said “All of these things are what I love in new work I feel like Vortex has given it the incubator that it needed to grow and be ready to fly.” A sense of home and room for experimentation has not only made The Vortex a brave space for Thompson’s work but also for members of the show’s ensemble One of the perennial performers in the Vortex family is Hayley Armstrong she’s the sassy alter-ego of multiple characters in Black Feminist Guide as when she snaps at Bea and Cee Cee and proudly shouts “Don’t let anyone tell you that you’re not a precious vessel!” got her start at The Vortex when she was a 15-year-old participant in the free summer youth theatre program They gave me my confidence and after high school I auditioned for plays and I was cast every year after that a Duke University candidate for the MFA in Dance as an Embodied Interdisciplinary Praxis plays Cee Cee (the body) in the ensemble and also serves as the production’s movement director Jones said that the movement for the piece is influenced not only by theatremaking and contemporary culture but also the healing arts Musical accompaniment is inspired by Africa and the African diaspora—jazz and beyond—and includes three original songs co-written by Thompson and fellow artist/scholar Guthrie R is also an academic in real life who teaches acting and movement at Texas State University Mozon marvels at the purpose-driven intentionality of Black Feminist Guide “There are wonderful explorations and liberties the playwright has taken with the theatrical conventions and with the music and movement She is glad that her work provides a way for fellow Black feminist artists to express themselves “Black women have to navigate the world and different institutions very differently than white women,” while also noting that her work appeals to a universal audience of women and men Navigating their way together is at the core of the partnership between The Vortex and The Black Feminist Guide to the Human Body it affirms her belief in the power of theatre to “shift the culture in transformative ways” and in her pursuit of “risk Ramona Harper (she/her) is a theatre and dance critic currently based in Dallas. She was formerly a staff writer for DC Theater Arts in Washington, D.C. Follow Ramona on Onstagentx.com Support American Theatre: a just and thriving theatre ecology begins with information for all. Please join us in this mission by joining TCG which entitles you to copies of our quarterly print magazine and helps support a long legacy of quality nonprofit arts journalism ©2025 Theatre Communications Group Each gift is a stitch in the tapestry that celebrates our resilience Donate to TCG! Jeff Mausbach didn't expect to end up in the wine industry And he definitely didn't expect to spend his life savings on a weed-ridden plot of dying grapevines in Argentina Mausbach seems a little crazed in the way that enthusiasts often do He has so much to say that it's hard to interrupt him He's talking about multiple projects at once: the highest-elevation vineyard in the world He and his viticulturist/winemaker partner Alejandro Sejanovich – who he calls "Colo" because of Sejanovich's red hair (it's an Argentine in-joke) – are making some of the most exciting wines in Argentina If you like complex terroir-driven red wines with a salty finish "Stems bring savoriness to the table," Mausbach said. "Malbec has the fresh fruit that we love There's a reason for everything Mausbach and Sejanovich do even if that reason is just to let the vineyard talk Example: Mausbach says the Malbec in Argentina is clonally different from Malbec currently grown in France because Malbec was brought to Argentina in the 1850s He says that post-phylloxera clonal Malbec selections in France were done for yield that's not a good reason to make 100 percent Malbec in Argentina He says most old Malbec vineyards in Argentina are field blends and that Sejanovich once had the job of "purifying" them by taking out non-Malbec vines and replacing them with Malbec it doesn't have the same complexity," Mausbach said How did he wind up in Mendoza building a restaurant and winery in the Central American style – one floor and sometimes one room at a time Mausbach was at University of Chicago when he got a job waiting tables at a trattoria He said employee meal was after service and "a bottle of wine was opened" He got the wine bug and spent 10 years in the restaurant business in Chicago first as a sommelier and then as a wine buyer When he moved to Buenos Aires in the early 1990s But he needed a job, so he sent his resume to every Argentine producer. At the time there was no real export market for Argentine wine. Only Nicolás Catena agreed to interview him. He became Catena exports' employee number two "When I started working for the family winery in the mid-1990s Jeff was my closest collaborator," Laura Catena told Wine-Searcher "He also worked very closely with my father because my father would ask him to write all his letters and emails Jeff has a very analytical brain and he is a wonderful person to collaborate with and to create new projects with." Mausbach met his future business partner Sejanovich who Laura Catena says was responsible for supervising the planting of many of Catena's best-known vineyards In 2009 they made one barrel of wine together from an old-vine Malbec-based field-blend vineyard Without even being sure they could sell it in 2010 they decided to go out on their own "I tried very hard to convince Jeff to stay at Catena but his heart was set on starting his own business – which obviously was a good idea given his success," Laura Catena said "I remember saying to my father how bummed I was that Jeff had left To which my father responded: 'When good people leave to go to a competitor you should be very upset and figure out what you did wrong But when they leave because they are going to start their own business one of Mausbach and Sejanovich's Cabernet Sauvignons made Wine Spectator's Top 100 There are other accolades that show that these are special wines from special places But you need a trained eye to see that the places are special where Sejanovich made their first barrel of wine from and where their Teho brand wines now come from It's distant from the alluvial fan that makes the soils at their other main vineyard It's not the highest-elevation vineyard in the world (Huichara in Jujuy province is The background is awesome like many places in Mendoza overlooked by the snow-capped Andes Mountains But Tomal Vineyard looked like nothing special to me "I was in the US trying to sell our first wines and Alejandro called me and said 'It's for sale'," Mausbach said He wanted us to spend all our money to buy it I said: 'That's all the money I have in the world You want me to use my life savings.' Alejandro said: 'I know Mausbach said he would call "the boss" – his wife Veronica "She came out and saw the beautiful landscape and said: 'This place is beautiful I was surprised by the vineyard I felt like I had been conned into buying I thought I needed a machete to walk through it We can bring this vineyard back to life.'" I can't help thinking about Charlie Brown's Christmas tree They took out all the poles that created trellising which they did using a system nearly unique to Argentina: burying a bud underground from the mother plant next to it "We had to do this process 8000 times," Mausbach said when Tim Atkin called Teho Malbec the best Argentine Malbec of the year." They keep plowing their returns back into the business They rented out winery space for several years before buying three hectares in the industrial city of Maipú to build on Have you been to Mexico and noticed people building houses one floor at a time with rebar sticking out of the top for the next time they have money to add a floor That's what their Mil Suelos winery is like They started with a single cement fermentation room and now they have 1.5 million liters of capacity They started a restaurant next to it and they're building a hotel But the winemaking work can't wait until the construction work is done Mausbach has an unusual theory about barrel rooms "This is where the winemaking happens," he said Here is where the wine is made." They have a winemaker just to supervise the barrels "Every one of these barrels is tasted every day." Trying to sell three different brands – Zaha for wines from Toko Vineyard but it's the name of the still-growing winery – isn't challenging enough So for their very high-elevation wines from vineyards in Argentina's remote north they named two of them Almacén de la Quebrada but I was busy at the time writing down some other interesting thing that he said Here are the wines I liked best from their portfolio 2020 Almacén de la Quebrada Salta Cachí You might think "Salta" is the description instead of the region Even from a winery that does stem inclusion to get this effect this is one of the saltier red wines you'll ever taste The red berries don't really come through until the midpalate but they do linger through the finish Made from 100 percent high-elevation Malbec 2020 Almacén de la Quebrada Salta Pucará Salted blackberries with a pretty perfumey note on the nose. Opens salty and stays salty through the finish. Salta, indeed.2020 Cielo Arriba Huichaira Vineyard Jujuy red blend This wine is from one of the highest vineyards in the world at 2700 meters elevation in Argentina's wild with an aroma that leaps at you with dark fruit and a notable beefy note and a juicy dark fruit character on the palate with a background saltiness that increases on the finish Yet it's not weighty: just 13.6 percent alcohol 2019 Teho El Corte Tomal Vineyard La Consulta Uco Valley red blend Made from 60 percent Malbec co-fermented with Cabernet Sauvignon Pretty aroma of black and red berries with black figs and a hint of tar Similar flavors with smooth tannins and a salty finish It's not herbaceous like Cab Francs from the Loire or simply "the lighter Cabernet" like those from Napa This very lively wine with bright red cherry fruit and a slightly stony finish will remind you most of Saint-Émilion 2020 Zaha Toko Vineyard Paraje Altamira Uco Valley Cabernet Sauvignon In 2021 the 2018 vintage of this wine became Zaha's first wine in Wine Spectator's Top 100 "And best for us it that it wasn't a Malbec it was a Cabernet Sauvignon," Mausbach said "Most California Cabernets have vacated this price point but the quality is not what it used to be." This is an elegant wine with fresh dark cherry 2020 Zaha Toko Vineyard Paraje Altamira Uco Valley Malbec Made from 90 percent Malbec co-fermented with Cabernet Franc and Petit Verdot with red fruit and notes of fresh flowers and herbs it has a salted raspberry flavor that gets saltier on the finish which is the hallmark of all of Jeff and Alejandro's wines Our latest update from the Bordeaux En Primeur front line features dry and sweet whites and a curious red Graves vintage The latest sales figures are bad news for producers hoping high-end wines will get them through these tough economic times It's all about music as much as wine as we round up this week's news from the wine world We conclude our search for the world's most sought-after wines with our overall top 10 Joe Biden might not be in the White House any more but his influence is still being felt at one crucial committee The science keep piling up: wine is good for your health Bordeaux En Primeur's Uncertain Start The En Primeur campaign for the 2024 Bordeaux wines has taken its first Ever wished you could hypnotize your friends into drinking better wine As traditional wine markets tighten and contract perhaps it's time to look at a previously overlooked wine market the death of Pope Francis was more than just another world leader's passing the Northwest Passage and the Transpolar Sea Route With the beginning of fall just two days away the sun at the North Pole is about to dip below the horizon for the next six months and the Arctic summer For the first time since the beginning of satellite measurements not only the Northern Sea Route (NSR) but also all channels along the Northwest Passage (NWP) appeared to be virtually ice free for an extended period of time Shipping traffic along the NSR is expected to double this year due to increases in the size and frequency of ships traveling along the route. In 2010 the Danish 40,000 ton MV Nordic Barents was the first non-Russian bulk carrier to use the NSR as a transit trade route Summer 2011 also saw the first supertanker the 160,000 ton Suezmax-class Vladimir Tihkonov While sustained sailing speeds along the NSR do not yet rival those along the world’s major shipping routes they are likely to continue to increase as first year drift ice becomes less likely to present a serious obstacle during the summer months The Vladimir Tihkonov maintained an average speed of 14 knots and sailed from Novaya Zemlya to the Bering Strait in seven and a half days surpassing the record set by the 74,000 ton Panamamax-class STI Heritage earlier this year Growing economic activity in the Arctic invites questions about the medium- and long-term prospects of shipping along the NSR represent a commercially viable alternative to traditional shipping routes What are the crucial variables in predicting the future of Arctic shipping Shipping companies and supporters of increased traffic in the region cite significant cost savings for ships that have sailed along the NSR and predict a rapid growth of Arctic shipping from 2 million tons today to 20 million tons by 2020 and oil and gas volume is predicted to grow along similar lines to 40 million tons per year by the end of the decade remain skeptical about the commercial viability of the NSR Canadian and American maritime experts say two percent of global shipping could be diverted to the Arctic by 2030 Experts cite a number of factors which may determine the future growth of shipping in the Arctic This series about the Northern Sea Route will explore how global trade dynamics and world trade patterns Russia’s Arctic natural resources development South Korea and Japan as Arctic maritime nations By examining the individual factors this series hopes to provide a framework for understanding whether the NSR will develop into a “Golden Waterway” or will remain a limited and seasonal trade route Growing economic activity in the Arctic invites questions about the medium- and long-term prospects of shipping along the Northern Sea Route (NSR) Part 2 looks at global trade dynamics and explains if the NSR would be compatible with world trade patterns “Arctic Shipping: Commercial Viability of Arctic Sea Routes” (MSc The actual sailing distance between Yokohama in Japan to Rotterdam in the Netherlands is roughly 20,000 kilometers passing through the Suez Canal but less than 9,000 kilometers via the NSR The majority of cargo ships that ply the world’s oceans operate on regular schedules follow a set route calling at a number of ports to load and unload cargo The global maritime industry operates on just-in-time cargo deliveries The ability to schedule journeys long-time in advance and to guarantee uninterrupted service are key for container ship operators The lack of schedule reliability along Arctic shipping routes represents a major obstacle to developing the NSR The Arctic Ocean off the coast of Northern Russia may be ice free anywhere from late June until November During some years the ice recedes early during the season and does not return until late into Fall while in other years the ice-free period may be as short as six weeks there are no guarantees when ice-free conditions start or end throughout the summer drift ice originating further north is likely to be pushed into the shipping lanes by wind and ocean currents Even during the summer months Arctic weather remains unstable and violent winds may interrupt the pace of regular liner services Between Murmansk and the Bering Strait the NSR passes along 2500 miles of nearly uninhabited Siberian tundra The lack of infrastructure and suitable ports along the route renders ships unable to receive timely assistance in case of mechanical breakdowns or damage Operating in such remote regions under harsh climatic conditions naturally translates into higher insurance premiums for cargo ship operators The world’s major container lines optimize their routes along a network of ports that offer developed communication lines into the hinterlands to distribute goods to customers and consumers As the NSR passes through mostly uninhabited territory no such stopovers are possible strongly reducing the route’s attractiveness for regular liner service operators such as the iron ore carrier Sanko Odyssey follow less predictable schedules and their routes depend more on changing supply and demand of less time sensitive items The NSR may also benefit from Russia’s oil and natural gas developments in the Arctic As the tundra’s permafrost begins to thaw the construction of pipelines and railways will prove ever more challenging and hydrocarbon resources may increasingly be exported via the NSR The NSR offers significant distance savings between Europe and Asia but scheduling uncertainty due to the Arctic environment and the lack of infrastructure in the hinterland will prevent the route from becoming popular with liner services the route may increasingly represent an alternative to more traditional shipping routes Part 2 of the series on the future of the Northern Sea Route discussed global trade dynamics and explained if the NSR would in fact be compatible with world trade patterns Part 3 will take a closer look at climate change in the Arctic and its impact on the future of the NSR How quickly do scientists expect the remaining summer ice to disappear and at what stage could year-round operations along the NSR commence which has had perennial ice cover for the past 700,000 years It is measured on a scale from zero (no reflecting power) to one (perfect reflection) depending on how “dirty” and old the ice is The water of the Arctic Ocean has an albedo of only 0.07 and thus absorbs the vast majority of the solar radiation instead of reflecting it back first-year ice is more likely to melt during the summer months than multiyear ice since ice that survives the summer is able to harden and become denser during the following winter The Arctic has witnessed rapid loss in multiyear ice: whereas in 1988 the vast majority of ice was between four and 10 years old by 2005 the majority of ice was less than four years old and others; Intergovernmental Panel on Climate Change Working Group I “Arctic Largely Ice Free in Summer Within Ten Years?” October 15 The NSR cannot be thought of as one clearly defined linear route but should instead be understood as the whole sea area north of Russia Due to highly variable and difficult ice conditions along most of the NSR the optimal route choice for vessels navigating the NSR will vary These navigational challenges throughout the Arctic have prompted the American defense contractor and industrial corporation Raytheon to develop an Arctic Monitoring and Prediction program (RAMP) and rapidly share data [..] which provides operational users key information in real time on critical topics such as ocean currents Thus far ships only travel along the coastal NSR and stay within 120 miles of the shore They must also pass to the south of numerous islands in the Laptev and Kara Sea as ice continues to exist further north throughout the summer the costal NSR has significant draught and beam restrictions one of the often cited advantages of the NSR will only materialize once areas to the north become ice free as well ships that are too large to pass through the Panama and the Suez Canal such as most Very Large (VLCC) and Ultra Large Crude Carrier (ULCC) Even then ships traveling along the route would still require ice-breaker assistance during the rest of the year and year-round operations cannot be guaranteed underestimate the speed at which the route is opening up The 2011 shipping season along the NSR is expected to last almost 4 months Once ice free and light ice conditions can be more or less guaranteed for several months the route may become more attractive for shipping owners Shipping companies are increasingly using moderately ice-strengthened vessels which can operate earlier during the summer and later into the fall The use of ice-strengthened vessels may eventually prompt Russia to reduce its ice breaker fees or abolish the escorts all together This would reduce costs and improve competitiveness for shipping along the NSR While shipping traffic along the NSR may soon be technically feasible year round ice will continue to hamper large-scale operations over the coming decades As long as the northern sections of the NSR remain choked with ice VLCC ULCC and Capesize ships will not venture into the Arctic but instead continue to travel along the traditional shipping routes Part 3 of the series on the future of the Northern Sea Route (NSR) took a closer look at climate change in the Arctic and its impact on the future of the shipping in the High North Part 4 will analyze the potential for cost savings along the NSR and discusses how credible these claims are Reliable figures about actual cost savings along the NSR are limited since less than two dozen commercial vessels traversed the NSR since 2010 Cost savings along the NSR are closely linked to savings in fuel costs Shipping operators can achieve fuel cost savings in two ways: A vessel traveling from Murmansk to Yokohama via the NSR will arrive at its destination seven days earlier than the same vessel sailing through the Suez Canal Due to the shorter sailing distance the shipping operator realizes fuel cost savings The operator also derives savings from the reduced number of days at sea which allows the the ship to make more return trips within a given time period resulting in increased revenue and potentially greater profits operators can also adopt super-slow sailing which more than doubles fuel efficiency a ship going from Murmansk to Yokohama can reduce its speed by 40 percent and still arrive in Japan at the same time as a ship sailing at full speed traveling through the Suez Canal Especially for bulk shipping operators transporting low-value raw materials the primary incentive to travel along the NSR may not be the reduced lead time He further explained that the costs for ice-breaker services amounted to $210,000 which he stated were comparable to transit fees for the Suez Canal Sovcomflot’s Deputy General Director Igor Pankov acknowledges that fees for the Suez Canal are less than ice-breaker assistance fees but states that “when time and fuel savings are taken into account the picture changes.” In addition he expects transit and ice-breaker fees to come down once more ships start sailing the NSR Thus far only small and specialized operators have achieved cost savings along the NSR How likely is it that these cost savings will materialize for a greater range of operators in the future Cost savings are closely linked to the origin-destination pair. While a trip from Murmansk to Yokohama is significantly shorter (by 7 days) along the NSR, a trip to Shanghai is not (2 days).16)Joshua No “Northern Sea Route may not be as viable as Russia claims” it is actually shorter to sail through the Suez Canal only goods that are shipped from point to point and along certain routes pairs will benefit from shorter distances the costs of using the NSR are frequently understated or ignored Besides the frequently cited costs for ice-breaker escorts shipping companies also incur significant indirect costs In order to sail the NSR operators have to file for a permit with the Administration of the NSR four months in advance Few operators are able or willing to plan that far in advance and deal with the bureaucracy of obtaining a permit the process of sailing through the Suez Canal only requires a 48-hour advance notice Shipping operators also face significantly higher insurance premiums when sailing through the hostile Arctic environment Navigating the NSR requires significant experience due to a dearth of accurate charts and the unavailability of the standard global positioning system (GPS) in high latitudes In sum these costs severely restrict the profit that can be materialized along the NSR A recent study has shown that a 50 percent reduction in ice-breaker fees would be required to make liner service between Rotterdam and Yokohama profitable profitability will for the foreseeable future be limited to a small number of specialized bulk carriers traveling along certain point to point routes Part 4 of the series on the future of the Northern Sea Route (NSR) analyzed the potential for cost savings along the NSR and discussed how credible these claims are The final part in this series will discuss China’s future as an Arctic Maritime Nation and take a closer look at its influence on the development of the NSR China has been very cautious about formulating and propagating its interests in the Arctic and continues to quietly advocate for unobstructed access to the region China’s advocacy for unimpeded access to the region for all states while certainly economically and politically self-motivated may help it to garner significant support from the international community especially smaller export- and import-dependent countries who would benefit from liberal access to the Arctic’s shipping routes especially its role as a growing exporter to Europe and the United States may allow it to overcome its position of weakness in Arctic affairs The country is neither an Arctic littoral state nor an observing member to the Arctic Council Chinese Arctic research remains focused on the environmental impact of climate change in the region; economic China’s economic development is highly dependent on international shipping and foreign trade contributes 46 percent to the country’s GDP China’s efforts to develop into an Arctic maritime nation and become a destination for Russia’s Arctic hydrocarbon resources are well documented In addition to commissioning a number of ice breakers the country continues to invest in ice-strengthened bulk carriers and tankers with dual-directional technology which combine the fuel-efficient bow of a cargo ship on one end and with the hardened bow of an icebreaker on the other Russia’s GDP is closely tied to its Arctic natural resource development which in turn depends on the ability to deliver these resources to the global markets Russia is seeking to tap Asian demand for oil and gas to help justify developing remote deposits in the Arctic and eastern Siberia Russia has a strong interest in developing the NSR into a commercially viable shipping route and ensuring access to one of the fastest growing consumer market for hydrocarbon resources: China and greater south-east Asia China’s demand for crude oil is slated to grow from 8.2 million barrels per day (mbd) in 2008 to 17 mbd 2030. Over the same period the production deficit of Asia as a whole will increase from 15 mbd to 48 mbd.22)Based on projections by Ramón Espinasa from the class “Energy Security: Western Hemisphere” at Georgetown University China is not only attempting to secure access to Africa’s growing share of world petroleum exports in order to satisfy its growing demand for hydrocarbon resources but also diversify the trade routes of its natural gas and oil imports Becoming an Arctic maritime shipping nation represents an important step in China’s goal to achieve long-term energy security The series on The Future of the Northern Sea Route looked at a number of factors which determine if the route will develop into a “Golden Waterway” or remain a niche trade route world trade patterns and global trade dynamics the economics of shipping lanes and the potential for cost savings and China’s role as a potential key benefactor of Arctic shipping are key variables to the development of the NSR as global shipping route References[+] The Arctic Institute is a 501(c)3 tax exempt nonprofit organisation with a network of researchers across the world LEARN MORE ABOUT US The Arctic Institute’s research and capacity building projects help make the Arctic a more secure Donate Get a weekly rundown of the Arctic’s top stories by subscribingto the Institute’s newsletter:The Arctic This Week Subscribe New to The Nation? 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Activate your online access Vince Staples’s self-titled Netflix show is a probing look at celebrity culture and the pitfalls of being only kind of famous Vince Staples as Vince in episode 104 of The Vince Staples Show the rapper is the self-assured griot of Long Beach The swaggering single “Norf Norf,” one of his signature songs captures his cutting directness and acerbic wit condemning the allure of street rap while boasting his hood ties His music celebrates the thrills of lucidity relishing the power and relief that follow from speaking candidly Staples brings that sensibility to his latest foray into television an eponymous Netflix show in which he plays a version of himself navigating a fictionalized Long Beach that’s exceptionally wacky and dangerous yet also down-home Nearly every episode of the surrealist comedy contains the question “Who is Vince Staples?,” but the show is more often a tableau than a study of its star Its droll jokes and cartoonish violence constantly give way to nuanced depictions of Black people a Black bank robber calls his white hostages uncultured for not recognizing Staples a proud Black mother declines money from Staples after he overhears her confessing financial woes to her kid These characters aren’t just vehicles for jokes; they are neighbors and nemeses with their own lives and agendas cocreated by Staples and Entergalactic writers Ian Edelman and Maurice Williams doesn’t get to dive deeply into this rich world The tight runtime leaves little room for subplots or a regular supporting cast so the show lacks the continuity of a traditional sitcom anchored in a workplace and situations to make the coastal city feel lived in Staples could never go Hollywood; he was raised 30 miles outside of it The show enjoys playing on Staples’s relatively low profile His meager fame agitates this snow globe of a series subverting and complicating the strange situations he stumbles into and out of When he’s pulled over by a cop in the first episode the dispatchers who run his plates struggle to identify him “I think it’s the guy from Abbott Elementary,” one says which references Staples’s real-life role on the ABC sitcom is doubly funny if you know that the show features two love interests played by actors who are rappers where he is recognized by the guards and other detainees They solicit concert tickets and try to network with him despite his being locked up like any other person the automated jail-phone system mistakenly records his name as “Nigga.” Amid the stumbles an inmate named Big Poke threatens to shank Staples his motive chillingly succinct: “You one of them niggas,” he tells Staples Staples finally gets released without incident but that mix of the mundane and the mortal lingers throughout the series: His upward mobility is tenuous Staples plays the straight man throughout these misadventures his composure contrasting with the opportunism or contempt that his presence kindles in others and The Boondocks all come to mind as the show swings between social commentary and slapstick action but The Vince Staples Show feels most in conversation with movies about Black California and criminals The episode “Brown Family,” which takes place at a raucous family reunion evokes the fragile escapism of the cookout scene in John Singleton’s Poetic Justice “Black Business,” set in a bank that gets robbed after Staples is denied a loan references multiple crime flicks—Set It Off Inside Man—to highlight the racial and class connotations between “heists” and “robberies.” Like Cord Jefferson’s satire American Fiction The Vince Staples Show is deeply conscious of the history of Black people being associated with crime and depravity unpacking assumptions about what it means to be a criminal as well as who gets to draw such distinctions In “Black Business,” not only are the bank robbers friends of Staples but they also take pride in their work They are far more likable than the haughty bank employees who dismiss Staples at the beginning of the episode Though the series ultimately feels like a tease it’s one of the most distinctive shows in recent memory to probe celebrity culture Staples isn’t interested in toasting fame or mocking the people who covet it He’s drawn to fame’s social effects—the ways it instigates new behaviors in everyone it touches The characters of the series aren’t just hangers-on “Who is Vince Staples?” is the wrong question The real riddle is: Who isn’t Vince Staples his contested attempts to just live are universal Stephen Kearse is a contributing writer for The Nation ShareSaveLeadership EntrepreneursTo Scale Your Business, You Must Embrace A NicheByKate Harrison A view looking down into the Grand Central Market in historic downtown Los Angeles [+] find a large variety of food stands and stalls selling groceries and other food related items Almost every thought-leader and hotshot CEO who made it big did so by focusing on one crucial aspect of their business: they embraced their niche Whether you’re a young upstart launching a new blog or an ambitious entrepreneur growing an online business or startup Judge Graham calls the opposite tactic “going an inch-wide and a mile deep.” Avoid this has become something of a personal mantra for him through a decades-long career as a digital marketing professional “I’ve learned the hard way through a few low lows that only by homing in on a niche can you be unique and stand out within your industry,” Graham said “You simply cannot be a generalist anymore.” Graham says “owning your niche” immediately gives you a leg-up on other businesses in the space He argues that businesses that serve a well-defined niche within an industry are strategically poised to demand a larger multiple “Don’t let your revolutionary idea die a quick death and end up in the overpopulated failed businesses cemetery,” said Graham By deciding early on to choose and target a market niche you take the biggest step towards positioning your company for scalable success and leadership.” Truly whittling down your ideas seems like a daunting task Here are some of his key strategies to finding a business niche early that will help you break out when it counts most A niche targets a distinct segment of the market you shouldn’t lose sight of what got you excited to start a business in the first place no matter how nebulous your thought process seems at first,” said Graham “You have to ask yourself where your experience lies what areas do other people remind you that you excel at and what kinds of things do you love to do in your free time even when you know you won’t get paid for it?” It might seem counterintuitive when thinking about drilling down deep but you have to start thinking broadly if you want to recognize your purpose This process lets you focus on what’s easy and what’s right in front of you — not the future glory from your imaginary business empire Going with your passion gives your mind room to hit that “sweet spot” when deciding your niche Once you have conceptualized the type of business you want Graham says you have to nail down the industry This step forces you to find a market for the niche that will actually pay you You don’t want your idea — whatever it is — to always just be a hobby You don’t want to abandon your shot at finding your potentially lucrative work “If you recognize the trends you’re good at in X but your immediate competition only serves X and Y you should intentionally put pressure on yourself to choose the right niche A little conceptual paralysis can be a good thing because it can force you to the edge of your comfort zone Graham says this is the space where truly revolutionary niche ideas are born meaning your hyper-focused venture will have a better chance to succeed Enabling bold ideas also gives you an edge up on potential competition “You can easily make note of other products that aren’t as good as your own and your profitable niche should naturally emerge if it isn’t already self-evident,” Graham said you can hop to a more thorough analysis that sets you up to beat your competition.” We live in an age where expertise is highly prized Having enough insight to take hold of those little-serviced needs means you’ll be able to establish yourself within that section of relevance to a particular problem or audience By analyzing your personal skill set and confirming your expertise you gain the knowledge to focus on a niche that could spark a seriously successful business venture. Start your free trial with Shopify today—then use these resources to guide you through every step of the process. Jason Wong is a serial entrepreneur who loves to thoroughly research and challenge himself to launch quickly and efficiently. In this episode of Shopify Masters, we speak with Jason Wong on why he decided to launch Doe Lashes, the research he did to find customer pain points, and how he launched with around $500 and in a matter of few a days. Jason Wong is a serial entrepreneur who loves to thoroughly research and challenge himself to launch quickly and efficiently. In this episode of Shopify Masters, we speak with Jason Wong on why he decided to launch Doe Lashes the research he did to find customer pain points and how he launched with around $500 and in a matter of few a days. Yotpo ( Shopify app), Loox (Shopify app), Okendo (Shopify app) Felix: What inspired you to start Doe Lashes? Jason: I was actually listening to a podcast. The really funny thing about where we're at now is that I heard on a podcast about two guys starting a lash brand and they're doing dropshipping They were using a drop shipping model on Shopify selling things using their Facebook ads expertise and were able to generate a lot of revenue After listening to the podcast I said to myself and maybe I could do it my way." I don't do dropshipping I've always been a person who has developed brands inventory around stuff and then ship it out on our own I was trying to see if I was able to get into the same lash market but doing it with a model that I was familiar with So I spent some time developing the product I set up some appointments to meet with vendors there too just so I can at least get an idea of what I was getting myself into So that's what the origin of the brand came from would you normally set up appointments with vendors this early? Jason: It was purely because I was on the way already All our factories are in Asia and if you're building a business in America it wouldn't make sense for you to fly to the vendor every single time you get a new idea But I think it was just because I was on the way for a vendor appointment for my other brands But generally what I would do is that every time I get an idea I do a lot of internal research on whether or not the idea's viable first Some products do require different certifications But just because I was on the way already and it wasn't really out of the way for me to visit these vendors I thought I would just set up the appointment Felix: What is involved when you are doing that kind of personal research to figure out if an online business idea is viable or not Jason: Oftentimes what I found is that we're really not the first person we're sometimes the fifth or sixth person to think of the same business idea the first thing I like to do is to see if there's any history of that product If someone has tried to create something and if so is it because the market wasn't ready for it Or was it because the vendor just wasn't able to supply what we envisioned So I like to see if other people have tried the idea first And what I like to tell everyone is that even though someone has taken the idea already It just means that maybe the market is validated and it is a sign for you to actually put more effort into it So the way that I like to see is to see what other people have done already and see what I'm able to do differently or if there's a roadblock that they experienced I like to see if I was able to circumvent that or if I need to stop the project entirely Felix: Depending on the reason for the previous failure how does that go into your decision on whether you should go in or not? Jason: There are usually a few reasons why I'll put a complete stop to it and there are a few reasons why I would actually put more effort into it Things that would cause me to put a complete stop are if I see that the product does not meet certification standards a couple of years ago I made a product called the Ramen Noodle Condoms which was really just what the name implies It was condoms that look like ramen noodles packets and they're a great product But what I didn't realize is that condoms are actually a type II medical device meaning that I am legally not allowed to distribute them or import them into the United States So I found out that part a lot later than I was supposed to and on the way to importing it into our country So things like that are due diligence that I was supposed to do before I put my time into that product I was now having a handful of products I cannot sell Some products require different certifications you need to go through different agencies for that any legal process I have to go through with this product if there are any vendors that make something like that already so that I can contact them to get any samples If you're creating an idea or a product that has never been done before in terms of the design and the functions it's really important for you to find something that's the closest to what that is Going to those factories in order to understand if this technology or the function or the product idea that I'm envisioning is even viable But oftentimes what would stop me is the legal process just because that takes a long time for me And the way that I like to structure my business is fast to market and fast to sell Oftentimes a lot of people get stuck in a particular stage of their business that ends up taking months or years So if I see something with huge legal roadblocks Felix: What did you discover when you started doing some more research into the existing lash market what's their favorite moisturizer; they can tell you a few brands that are really well known But what I found is after asking 20 to 30 people around my circle and then later on I asked more That was a very fascinating thing for me because I was essentially gauged from a market where not a single brand owned the majority of the market I was coming into a market competition where I was competing with a bunch of larger brands but they don't have that much power over me besides the fact that they have more money and resources meaning that I was able to attract new people to try our brand because there wasn't brand loyalty Felix: Is that what you consider as a certain green light It's one of the elements that I do really like personally because when you see a market where there's no large player but there's a lot of players what that tells you is that it's a validated market It's a market where there's a lot of people purchasing but not one single brand has been able to perfect the product to attract everyone So that tells me that there's an opportunity for me to create that solution for me to combine the elements of what makes each brand great Knowing that I was able to study all these brands and combine them together to create a single solution that may be able to attract the larger market and get bigger market share that was a green light for me but it wasn't the only thing that tells me to go for it Felix: What's your plan of attack once you know that there is no brand loyalty? Jason: I like to focus on functions and features rather than a brand A lot of bigger brands like to close off the existing brand value that they have already But obviously as a new player with not a lot of capital that wasn't an angle we can go for We try to look for pain points that people have with these products sometimes people will set issues with your product such as buying a particular type of moisturizer that ends up drying faster So I try to go through these other brands and understand what their consumers are complaining about And the common pain point is that when they put the lashes on In our ad copy and the way that we portray ourselves to the public we try to hit on those pain points and tell them that this product actually does not have the problems that people are experiencing with other brands we just like to present them that this is a solution to the problems that they may be having And that if they'd like to avoid having the same issues that they have with the other brands once you have a little bit more brand recognition You can do a little bit more types of angles our first step was to convince people that this product was superior to whatever they were buying already Felix: How do you know what function or features or maybe even what problems you should be building functions and features to address Jason: A lot of these come through study groups and surveys I have about 12,000 followers on just Instagram I asked people to enter information in exchange for a free product once I launch it So I asked them very detailed questions about what's their age what's their experience in using these products If they could change two things about this product So asking them very detailed questions to identify the pain points that they have After seeing what are some common pain points that these people have So that's pretty good sample size for what I was just looking for I saw four or five common pain points and I first looked at myself and I asked Is that something that I can actually change So that was the first direction that I had set for myself was to solve the pain points You want to make sure that your product looks good while solving the same problem So that's really where the time is spent is to bridge together the solution Felix: What's involved here in the design process to make sure you are solving the problems but then still keeping it aesthetically looking good? Jason: I'm not a person who had worked at false eyelashes in the past So I spent a lot of time studying beauty brands I think a common mistake that people make when looking at the design is that they like to limit themselves to what other people in their own space is doing you end up creating a second version of another person's brand What I like to do is I like to draw inspiration from other beauty brands such as moisturizers I remember sitting in my bathtub and looking at the copy from a Dove body wash bottle for about 10 minutes just reading how they write their words or how they use the color of the logo to use the same color on their text Just seeing inspirations from different designs and then trying and seeing if I was able to replicate that with my own brand just because I felt that Korean brands are able to convey colors and formatting a lot better than Western brands where it's a little more plain and straightforward that's why when you look at Dove it's very colorful it pops in your face and if you ever see it in retail shows I'd like to just draw inspiration from different mediums and see if I'm able to create something that people in my space have not been able to do Felix: How do you immerse yourself in a way where you actually are able to get actionable things that you can try to implement in the design of your product There's the external design and internal design A lot of times internal design is what creates a function of it And I think that was one point that I did not mention previously I just purchase a lot of products in the space that is similar to ours and try to see how they're able to structure their designs together to create the final product So that's things that you can see on the outside: colors purchase as many things as I can that I feel like could add to the final study of what I want my product to be and now we're getting into the product design is what creates the product with the function that you envision So I went out and I bought about 20 different pairs of lashes from 20 different brands and I study how they make their cotton band whether the thickness contributes to durability and comfort And I found that there needs to be a balance between having a thick enough band to hold the lashes and a thin enough band to be comfortable So I have to purchase a bunch of lashes and test out which one was the best for the band And then I go into the lash hair themselves I look at what type of materials I can customize whether it is nylon or whether it is plastic There's a lot more complicated stuff beyond that but that's a general idea that I like to take elements of what I like and essentially Frankenstein my own product at the end that draws inspiration in elements that I think make other products great Felix: How did you know what was actually possible to create in the real world Jason: I own a lot of this to my friends and my girlfriend who has been great in advising me your online presence will be your greatest resource I went on beauty forums and I read as many posts about lashes as I could Literally every single thing that I got access to Just to understand the fundamentals of how it works So I saw that some people were actually stacking two pairs of lashes on top of each other to make them fuller because a lot of the lashes on the market at the time didn't offer that type of style what if I just made a style where it's two pairs of lashes stack together I asked a lot of my friends who were already wearing lashes I often set up impromptu study groups where I just give them products that I've made I just want to really emphasize on the good questions park A lot of people ask questions to their friends and they ask stuff like "What do you like about it?" Those are questions that won't really get you anywhere because a lot of times people will tell you they like it just because they're your friend What I like to do is ask very specific questions "If you were to wear these lashes for the entire day what would be some noticeable things on the lashes that you'd want to have?" So they're like "I want this to feel weightless on my eyes I want it to look natural." So we're able to create styles and products that fit that description just because we understood what people wanted by asking the right questions Felix: When are vendors involved as you are crafting your product Jason: At this stage I would like to get as many samples as I could from different suppliers and I try to understand what makes them better than the other What I like to do is I like to ask them about what makes their product stand out I don't try and pin them against other suppliers because then they're able to tell you exactly why they think their product will be superior So getting a lot of samples from different suppliers understanding what makes them different on the fundamental level made by hand and where's the assembly line I try to find out everything that I could about the product and find out online what makes them better or makes them different One funny thing was I did not know that cheap lashes were made in North Korea That's one thing that I found out when I was in China I visited several vendors after I already made purchase orders with one I just wanted to understand if there are better options if I could really improve my products And I found that for some factories their assembly line was in North Korea and they just sent it to China for the final packaging So that made me really uncomfortable because knowing the working conditions there I wasn't really comfortable with putting my products through that type of assembly line So I found a factory where they do everything from start to finish So I think doing my own due diligence in the type of people I like to work with was really important Even though getting the manufacturing in Korea would've been about 60 I just felt more comfortable knowing that our products were made in an assembly line in a factory where people have good working conditions Felix: Where do you think most entrepreneurs get stuck on Jason: A common mistake that I found is people getting stuck on things that really don't matter in the long run. I know some people who make a logo and a website design and they're stuck on that for two three months because they don't know what's the right combination Sometimes they just don't have time for it But a lot of people are getting stuck on things that aren't making them money immediately rather than focusing on the product I'll say what makes us able to get to the market so fast is our ability to understand what needs to be done first making the store and getting the marketing strategies out to get in front of people's eyes faster rather than getting stuck on things like making a Shopify store or creating a theme or like custom-design an entire theme When we start every single one of our brands And just through some simple designs on Upwork or Fiverr we're able to get the store up and running with amazing designs in about one to two days We like to expedite processes where we could we have a template where we just follow and create brands in about 48 hours Felix: What's involved in that template to start a brand in just 48 hours Having that high up on the list is that every single time we want to launch a new product or we launch a new store we'll follow that template and set requests for those things to be done so that when the store's finished So what we do is a lot of internal stuff like changing our upsell model But when we put the request in for the new logo so just our contractors and our freelance designers can work on those while we're working on the internal stuff So at the end of 48 hours or maybe the next day we're able to have all those things together and just launch a product So I like to launch and then improve later on It's a model that some people may question Just because I personally like to launch things really fast and fix issues as it comes Obviously I'm not going to launch an incomplete website but what I mean is that I like to launch the bareness as a city of something and then throughout the time when I feel like I could change something three days I realize that the email popup isn't really converting so I change the copy on it So a lot of times people are stuck on that stage before they even launch a website What text should I use?" I just like to launch it and see and optimize it from there We can see the stuff that we're improving after we launch a store aren't things that are going to make or break our website It's more so like we launch things that'll make us money immediately and then fix the things that will make us more money throughout the way Felix: What was the reason for setting a $500 limit on your launch budget? Jason: The reason why I got you this figure was because for my first product that went viral I only spent $500 and I was able to create huge sales for the brand I did about 200K in a week or so with just $500 and it's been about three years since I launched that book “Can I replicate that with the same amount of money?” And the answer is but I'm able to replicate the same level of success in our own way So I set that artificial ceiling for ourselves to use only $500 and try and recreate the same business model using the same framework that we set Doing so I was able to create the brand by focusing more on a product and then dedicating a lot of time to do things I'd otherwise hire people for A lot of people have product ideas and they hire someone to measure store and that will end up costing you $1,000 or more What we did was we did everything internally I spent most of the money on things that I cannot change even though we have our own warehouse now for our other products I decided to fulfill all the products within my own home So I was fulfilling things in my living room just like how another person with just $500 would do I started this challenge because I want to know that if I'm trying to teach people how to do this I should be able to do this over and over again I set up the same model and I was able to follow it to a tee and recreate the same brand Felix: What did you spend $500 on to basic kickstart a fourfold business And I order 50 units of each skew and I have four skew total So that's 200 units at about $2.70 per unit for the product I look at it sometimes and see how far we've come We can just create stores here and there without the trial thing And then I pay someone on Fiverr to create the first few designs for the banners and then I hired a friend to do our local for about a friend of ours So I only had to pay her a couple of hundred bucks it comes out to about $700 for the full brand with the designs done But the reason why I spent so much money on that design was because for this brand in particular you have to make sure that the packaging in a box looks really nice You don't have to spend a lot of money on the box if your product is good enough So that's just one thing that I want to emphasize we didn't go too far beyond our initial budget and were able to get the stuff to the market and be able to be sold at just $700 Felix: When should an entrepreneur know that they can now focus on stage two activities Jason: Everything that I do in stage two comes from being funded by stage one And this will be different for every single person What we found is that stage two is an ongoing process It's not like you just need to transition your entire brand into stage two The designer in South Korea should redesign our entire packaging because it was a design firm that designed the color So we had to make enough money in stage one to afford that you have to first generate enough revenue in stage one for you to afford that on top of your overhead expenses So that number will be different from person to person but typically you should be able to get to stage two after five to six months in business Felix: What is the marketing strategy that you have today to get customers Jason: Our strategy has always been an influence on marketing, and that was one thing that I focused on for my initial brand. I'm a firm believer that you should do things that you're good at and focus all your time on that. And if you need to expand outside of that, hire the right people for it. For us, we knew that our bread and butter were influencer marketing and following the same framework of marketing strategies that we have for our other brands I was able to replicate that for this brand And what I mean is that we focused a lot on micro-influencers We have a previous list of micro-influencers with under 100,000 followers that we had from our previous brand but you can easily recreate the same list on your own just by reaching out to people What we did was that we reach out to those people and reach out to their friends who may not have known us or our previous brand and ask them if they'd like to receive a product in exchange for promotion on their end But one thing that we did differently about the strategy was that we don't ask people to commit to stuff A common mistake that people do when they work with influencers is that they ask them to promote something in exchange for a product It sounds pretty aggressive because they're like You just have to post about us." And from a brand side but the influencer who's receiving the same message 100 times a day you're just another brand that wants something from them So the way that we approach influencers is that we don't ask them to commit to anything you send on a product as a gift and if they like it we are first able to improve our response rate from the influencers because they actually feel like they're comfortable with us Then two is that people who try a product and actually like it are willing to post about it You cannot send them a really bad product and expect them to post about it because that would decrease their integrity amongst their audience Always make sure that you're working with influencers who are within your niche You are working with people who you have developed good conversation and relationships with over the past few days that you've contacted them you just want to make sure that you're sending them a good product with no commitment because that's how you're going to get most of the responses Felix: What are you doing that time to establish some kind of relationship with them in just a few days before you ask them to get their feedback on a product Jason: I actually ask them the same questions that I ask my friends I'm sort of looking at them as influencers or celebrities or whatever I see them as ordinary people who are just content creators and have a good amount of audience What would you like to see different in the lash industry?" Asking the same question just to get them comfortable and get them to answer a question first and foremost Can we send you something to try?" It's a long process but it's the same process that we have followed over and over again And it's this process that a lot of people can replicate just simply because of how many micro-influencers they are on a market today following that same concept you're able to do the same thing for your product You just have to change the language a little bit the core message here is that you need to build relationships with your influencers or else you're just going to be left unread in their other inbox Felix: How do you identify which people are worth working with? Jason: The previous list that we had was for our clothing brand So we found that the same list could be applicable to the same brand But for most people who don't have that list Let's just pretend that I don't have that list I will look for people who are already working with other brands what type of beauty brands they're working with or what type of content they're creating And then I can determine whether or not they're up to promotions Oftentimes you want to look for content creators who are already tagging brands in their content and are very open to working with new brands so you can look at people who are working with brands who are not big And if they're willing to text small brands it means that they're more willing to work with brands like you it is always important to look for a lot of times you would think that you just need to send products to models that you're selling a bikini But what people don't realize is that people that follow models are oftentimes guys So you're essentially getting someone to sell your product to a bunch of guys it's really important to find the right influencers for that While influencer marketing may be very cheap and effective it takes a lot of time for you to understand what to look for And that takes months or years to fully master But if you're willing to put in the time for it you're able to vet each influencer that you're working with easily Learn more: It’s Your Time to Shine: How to Find and Work With Instagram Influencers in 2021 Felix: What do you think that you'd do differently about influencer marketing vs Felix: You have a page that says Love Letters, which are reviews, and then Track Order, and then Help. How did you determine what should go into the navigation? Felix: Any tools or apps that you'd recommend other people check out? Jason: For the review I use Okendo. It's a little bit pricey, so I don't recommend going to Okendo and we didn't go to Okendo in the beginning. We only went to Okendo in stage two, and stage one we just used more affordable types of reviews like Yotpo or Loox review sites. And then for Shop on Ig, I believe the call shop on Instagram. So those two apps are my favorite apps for this part. Felix: What has been like the biggest lesson that you've learned so far in building this brand that you want to apply in future brands or in the future of growing this brand? Whitefish Heliarc specializes in stainless steel hygienic welding Matt Young and Keanan Janus had an epiphany over a beer As professional welders and welding inspectors both men found themselves looking at a weld at the business they were patronizing and both had the urge to pursue a career outside of the company something they could build and call their own After seeing what they considered a poor welding job the men decided to start their own niche welding business which specializes in code-compliant sanitary TIG welding Their business’ niche specialty is hygienic welding on stainless steel for breweries and they’ve picked up enough business to fill up their weekends “We figured this was a way we could serve the community and do what we love,” Young said “I wanted to see if [this business] would work started in earnest in 1941 after Russell Meredith of Northrop Aircraft managed to perfect the technique He named the process Heliarc because it used helium in part It’s also known as gas tungsten arc welding Young and Janus both started welding when they were teenagers either because of a vocational class or because of family influence Navy and served from 2006 to 2012 working on hull maintenance The two began working as welders together at Applied Materials and they got to talking about what they’d like to see in their futures their niche welding service has proven to be popular Hygienic welding means it is sanitary and at food-grade levels Other types of welding can trap food and bacteria “is not good.” Whitefish Heliarc works only with stainless steel an alloy that can be tricky to weld because of its variable compositions “It’s a little more difficult to weld than other metals,” Janus said Young said working in California got him used to having more regulations on welding practices and he and Janus bring those rules to every job Both men are certified welding inspectors through the American Welding Society which helps them not only scrutinize the job they’re doing but also brings an understanding to complex standards and codes around welding They follow the guidance of the American Welding Society when it comes to code compliance “Every joint we put out or place anywhere is also inspected,” Janus said Whitefish Heliarc has had plenty to do with the existing local breweries with projects at Great Northern Brewing Company and Kalispell Brewing Company They also intend on taking their business on the road soon It’s still a side hustle to their everyday lives and Young and Janus still feel like they’ve got a nice balance in their lives they hope to find enough business to make this a full-time job and one that allows them to stay in the valley Young said the recent population boom in the Flathead is a sign that more breweries are likely on their way and believes Whitefish Heliarc will be able to find its feet if current trends continue but we’re balancing it well with working and fishing,” Young said For more information on Whitefish Heliarc, visit www.whitefishheliarc.com The continued support from our readers keeps our lights on and helps sustain local independent journalism in northwest Montana Please consider a one-time gift or sign up for a recurring contribution and join more than 500 readers in the Editor’s Club Click here to read about the impact the Beacon has on the community. © 2025 Flathead Beacon, All Rights Reserved. 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