professor of American studies; director of the Center for Leadership and Learning (CLTL); and a standup comedian leads the Skidmore community in laughter and learning The Skidmore College riding team finished in fifth place at the 2025 IHSA Nationals Map & Directions Sélection parmi les sorties de cette semaine • Young Gun Silver Fox : “Pleasure” (Légère) • James Harman : “The Bluesmoose Session” (New Shot) • Noir & Gerber : “Partout Partout” (Les Disques du Progrès)  • Jacques Schwarz-Bart – Gregory Privat : “22” (Buddham Jazz) • Bacao Rhythm & Steel Band : “Big Crown Vaults Vol • Luc-Hubert Séjor : “Miziki Filamonik – Spiritual Sound” (Heavenly Sweetness) / So you want to find the portal to a higher plane?” Such are the questions posed at the top of Eight Immortals the new song cycle from burgeoning composer/lyricists Sam Tsui and Casey Breves which delves into the Chinese folklore of the ethereal octet – archetypes each with their own story detailing how they attained it whether “guided by dreams and diets” or “tested by trials of self-denial and self-discovery.” which have long been woven into ancient myths reimagined by Tsui and Breves as a series of self-contained vignettes – culled from “the juiciest parts of their individual stories,” says Tsui — each updated with a contemporary twist Boasting an all-star cast of Broadway legends ponder) the question: “If they did all truly discover a way to escape death perhaps the paths they found are available to any of us we sat down with the dynamic duo – who are also a married couple in addition to their professional partnership – to discuss all things love from the origin of their partnership to the meaning of personal success and enlightenment to their social media success Embrace the beauty and wonder and strife, and scroll through their answers below. If any of their responses shift your perception of growth and personal transformation, well…. you’re well on your way to eternal life Can you speak a little bit about your professional history together Sam Tsui & Casey Breves: We first met at Yale recording and doing life together pretty much since then – we got married in 2016 and our daughter Elaia was born three years ago we’d like to think we have a pretty consistent and productive working relationship What was the inspiration behind pursuing this topic Tsui: I grew up with the stories of the Eight Immortals these fascinating legendary figures of Chinese mythology They have centuries of folktales and art associated with them… [my] dad had little statues of them in our living room each representing a cross-section of society — [meaning] young or old rich or poor — who attained immortality through a variety of crazy [methods] I’ve always dreamed of putting a theatrical spin on these folktales I grew up with What about it being a song cycle makes it easier for people to digest Tsui: There are so many great adaptations of Greek mythology in opera and musical theater but not a ton of East Asian mythological representation we landed on taking the elements of the mythology that spoke to us most [to] craft [this] song cycle and give the Eight Immortals the modern remaining we thought it deserved Breves: And we settled on a song cycle album as the first iteration of this piece because it seemed like the clearest way to tell the stories we wanted to explore… and then built a world around those stories which are then connected by narration that gives some mythological context and three ensemble numbers that hopefully elevate the individual threads into a grander and more timeless tapestry Tsui: And I think these themes of transcendence and transformation really lend themselves to musical theater Can you describe the process of adapting these folktales to music Was there a specific element of this folklore that drew you in Breves & Tsui: There’s such a wealth of juicy material with the folktales so I think we just absorbed everything we could and then tried to think of the most compelling and unexpected ways to reframe the story (or elements of the story) in a contemporary setting So, for example, we took the story of He XianGu, an ascetic who attained immortality through a strict adherence to a diet of crushed mica (rocks) and reimagined it as a sponsored post by a (possibly supplement-addicted) mommy vlogger sharing her morning routine to her followers Each story ended up speaking to us in a different way is there a particular story or song that speaks to you Breves: I relate to most to Lan Caihe, who wanders from village to village singing songs and carrying baskets of flowers Tsui: I love Zhang GuoLao, the eldest of the immortals, who was notoriously debaucherous and carefree I try to embrace that sort of outlook on aging (laughs) Do you think some of themes represented in these myths are relevant to our life today Why is it important that these stories be told Tsui: The themes in Eight Immortals have a lot of resonance for us for the listener… [including those of] personal transformation longing for moments of clarity and transcendence and striving to find the best way to live the mortal lives we have while hoping for something more I think many of us in 2024 are striving to “optimize” ourselves seeking physical and emotional upgrades and reinventions we did get to explore a bit of Daoist philosophy as well in writing the piece The “three treasures” of Daoism are compassion all of which the Eight Immortals must master though they might remain lofty goals for most of us mortals Breves: My understanding of enlightenment from exploring Daoism is being in harmony with the natural order Tsui: “Dao” 到 is a path or a way. I see enlightenment as a continual process toward that that natural harmony Casey mentioned. At the end of our cycle, we also explore the idea that our path toward enlightenment is never truly complete What’s one thing you would want to do if you were immortal like these characters Breves: Astral projection and body-swapping like Li Tieguai Tsui: Most of the immortals were skilled at alchemy - I’d love to be able to create gold from nothing so I could fund more great art! With over 3 million subscribers and more than a billion views on YouTube (in addition to your presence on TikTok and other platforms) you’ve both been extremely active and impactful within the social media music landscape how do you think these platforms have impacted the way people and projects are promoted and seen Tsui: I’ve been on the frontlines of digital music and content creation, beginning all the way back with [the creation of] my YouTube channel as a college student… I’m so grateful to have gathered a following of folks who connect with my music and story It’s been incredible to see the ways social media can give any artist direct access to a huge global audience but it can definitely also be a challenge to cut through the noise We’re very much still figuring out how a project like this – which we hope people will want to listen to in its entirety – fits into the confines of the short-form social media world what advice do you have for an average TikToker who may be similarly looking to make it in the musical theatre world Tsui: I’d say the biggest hurdle is getting over being too precious about any single piece of content (and this is something we still struggle with as perfectionists!) If you want the chance for any algorithm to decide to show your work to the world you have to start by just letting go and just posting in the first place Don’t be too attached to whether a single video or performance “blows up” or “goes viral” or not… [It’s] about consistently sharing stuff that you genuinely love and enjoy making Do you see a full-fledged production in the future Tsui & Breves: We are planning to present a concert version of the piece within the next year… and would absolutely love to see it staged in the future What do you hope listeners take away with them when they hear this song cycle What message do you hope to instill in them with this piece Breves: We hope listeners can come along on this journey with us and feel uplifted and transformed by the experience they’re entertained by the sillier elements but also moved by the universality of the themes the piece explores our incredible cast absolutely elevated this piece beyond our wildest dreams and we can’t wait for everyone to hear their incredible work Eight Immortals: A Song Cycle, with music and lyrics by Sam Tsui & Casey Breves, was released digitally on December 6, 2024 and is now available across all music streaming platforms. To stream the album in full, click here, or follow the musical on social media here Here at Tasting Table, we care a lot about coffee (this is a team of writers, after all). We're slugging back lattes and cappuccinos (which are not the same thing, for the record) while explaining why the "macchiato" on a Starbucks menu is not, in fact, a true macchiato This former-career-barista-turned-food-writer personally has a lot of opinions in the espresso sphere — including that if you order your breve lattes large a breve latte is not a beverage you're going to want to order large while half-and-half packs a comparatively formidable 10.5%-12% This is the reason why just a splash of half-and-half is all it takes to lighten black drip coffee while a more generous slug of whole milk is necessary to lighten coffee to the same degree This is an overwhelming amount of half-and-half to drink even if you aren't normally lactose intolerant but "breve" is literally the Italian word for "short," and ordering it long is a tall mistake Fans like the breve latte for its lush richness and absence of sweetener If you feel comfortable bodying a full two cups of half-and-half quickly regrettable java experience to you Some coffee fans even prefer to order their breve lattes cortado-sized to forgo some of that density this sumptuous bevy uses an equal ratio of half-and-half and espresso in which case the thickness of the dairy complements that espresso proportionately well there is a way to balance out a large breve latte without using quite so much half-and-half: Combine the breve latte with an Americano simply hot water topped with two shots of freshly-pulled espresso To translate this into a happy-medium "large" order ask your barista for "a long Americano topped with steamed half-and-half." You'll still get that rich and the hot water will extend the overall fluid ounce capacity for that big bev you need to get through a dragging weekday morning Also, happily for beginners at latte art, steamed half-and-half creates the ideal glossy, silky microfoam. It's easy to "paint" with thanks to its thickness and stark white opacity, making the breve-Americano a tasty way to practice your art and satisfy your caffeine threshold at the same time Broadway Off-Broadway Off-Off Broadway Cabaret Dance Opera Classical Music Minneapolis / St. Paul Connecticut Atlanta Chicago Los Angeles Central New York WEST END UK Regional Canada Australia / New Zealand Europe Asia Latin America Africa / Middle East TV/Movies Music The album is set to release on December 6th Social media sensations Sam Tsui and Casey Breves have unveiled their latest musical endeavor which draws inspiration from a series of popular Chinese myths This innovative work transforms traditional tales into a contemporary musical format showcasing the duo's unique blend of pop and musical theater 2024 and will consist of 22 tracks that offer listeners a fresh perspective on the legendary figures known as the 'Ba Xian' or 'Eight Immortals' 'Eight Immortals' features a collection of self-contained vignettes each song serving as a modern interpretation of one of the eight classic archetypes from Taoist legend The album's tracks range from the absurd to the heartfelt vocal-driven score that invites audiences on a transformative journey Included in the lineup are songs such as 'So You Want To Be Immortal' each providing a unique take on the themes of enlightenment and personal transformation a married songwriting duo based in Los Angeles have previously collaborated on various music projects but 'Eight Immortals' marks a significant milestone in their artistic journey With over 3 million subscribers and more than a billion views on Tsui's YouTube channel their combined social media presence has made a notable impact on the music landscape including 'Singing One Song From Every Country in the World' and ‘Musical Theatre Monday’ has further cemented their status as influential figures in the digital music community The duo's connection to Chinese culture is evident in the thematic exploration within 'Eight Immortals' This song cycle poses critical questions about the meaning of enlightenment in the modern world and the lengths individuals may go to achieve it The production of 'Eight Immortals' involved a thoughtful process where each song stands on its own while being intricately linked through scored monologues and reprises This structure enhances the listener's experience providing a mythic context that deepens appreciation for the timeless themes embedded in these ancient stories The album not only entertains but also encourages introspection about personal growth and transformation making it a relevant addition to the modern musical canon Dead Outlaw is the darkly hilarious and wildly inventive musical about the bizarre true story of outlaw-turned-corpse-turned-celebrity Elmer McCurdy. As Elmer’s body finds even more outlandish adventures in death than it could have ever hoped for in life, the show explores fame, failure, and the meaning – or, utter meaninglessness – of legacy. Dying is no reason to stop living life to its fullest.  The 40th Annual Lucille Lortel Awards – the only New York theatre award to exclusively honor Outstanding Achievement Off-Broadway – are being presented tonight, Sunday, May 4, 2025, at NYU Skirball. Check out live updates from the ceremony here! Tony-nominee Cole Escola, creator and star of Oh, Mary!, sat down with CBS Sunday Morning for a chat about their smash Broadway comedy, artistic origins, and why the riotous new work comes from a surprisingly personal place. Watch the segment and the extended video interview with Cole below! Check out title star Luke Brady and the company of Hercules bringing their gospel truth to the Britain's Got Talent stage. Watch the video of the cast performing some classic tunes from the film as well as a brand-new song for the mythic hero himself!  With Paul Mescal officially set to star in a 2027 production of A Whistle in the Dark—a co-production between London’s National Theatre and Dublin’s Abbey Theatre—industry chatter suggests the Abbey is now eyeing two more major Irish stars: Saoirse Ronan and Andrew Scott. function closestickysocial(){document.getElementById("foxsocial").style.display="none";}@media(max-width:1024px){.most-popular,.video-row{display:block;margin-top:25px}}Videos and exclusive discounts on tickets to your favorite shows © 2025 - Copyright Wisdom Digital Media, all rights reserved. Privacy Policy The duo's connection to Chinese culture is evident in the thematic exploration within 'Eight Immortals'. This song cycle poses critical questions about the meaning of enlightenment in the modern world and the lengths individuals may go to achieve it. Follow Sam Tsui and Casey Breves on Instagram at: __________________________________________________________________ Check out "The Roundtable with Robert Bannon" here on BPN on the daily, on YouTube, and on social media! For more info on Robert- www.RobertBannon.com © Broadway Podcast Network The page you are looking for cannot be found You may have followed a broken or outdated link the page you were looking for could not be found The most comprehensive sci-tech news coverage on the web • Jon Cleary : “The Bywater Sessions” (FHQ / Well Kept Secret) • Tim Gartland : “The Right Amount Of Funky” (Autopublié)  • Emma-Jean Thackeray : “Weirdo” (Warner Music UK) Sélection parmi les sorties de ce vendredi • Galactic and Irma Thomas : “Audience With The Queen” (Tchoup-Zilla/Thirty Tigers) • Ina Forsman : “After Dark Hour” (Jazzhaus) • Ann Nesby : “ANNiversary” (It’s Time Child) • Adja : “Golden Retrieve Her” (Sdban Ultra / N.E.W.S.) • Big Dave & The Dutchmen : “Big Dave & The Dutchmen” (Naked) • John Primer : “Gown In Mississippi” (Blues House Prod.) • Bootsy Collins : “Album Of The Year #1 Funkateer” (Bootzilla Prod.) • Andrew Duncanson : “California Trap” (Run It Back) • Janiva Magness : “Back For Me” (Blue Elan) • Jessie Reyez : “Paid In Memories” (Island) • Butcher Brown : “Letters From The Atlantic” (Concord Jazz) • The Sure Fire Soul Ensemble : “Gemini” (Colemine) • Joe Armon-Jones : “All The Quiet (Part I)” (Aquarii) • Kalisway : “Take Me Back (World Of Eras” (EP) (Kalisway LLC/Cloture) Scrapper’s Blues (Remastered 2025)” (Craft) • Sunny War : “Armageddon In A Summer Dress” (New West) • Max Hightower : “Nothin’ But The Truth” (MoMojo) • HeavyDrunk & Watermelon Slim : “Bluesland Theme Park” (4142 Music) • Candi Staton : “Back To My Roots” (Beracah)  • Durand Bernarr : “Bloom” (Dsing/Creat Music Group) • Kim Cruse : “Mixed Emotions” (Harmonix Factory) • “Strata-East: The Legacy Begins” (Mack Avenue) • Seth Walker : “Why The Worry” (Royal Potato Family)  • Johnny Rawls : “Make Them Dance” (Catfood) • Ronan One Man Band : “Piece Of Life” (Binaural Prod • A Bunch Of Birds : “A Bunch Of Birds” (Gutfeeling) • Jus’ Blues 25th Anniversary Legends Collection (Jus’ Blues Music Foundation) • Jordan Rakei : “The Loop (Deluxe)” (Decca) • Shawn Pittman : “My Journey” (Continental Record Services) • The Lewis Express With Chip Wickham : “Doo-Ha!” (ATA)  • ZZ Ward : “Liberation” (Sun Label Group) • Derya Yldirim & Grup Simsek : “Yarin Yoksa” (Big Crown) • Mereba : “The Breeze Grew A Fire” (Secretly Canadian)  • The Altons : “Heartache In Room 14” (Daptone)  • Susana Baca : “Conjuros” (Pregón Producciones) • Jimmy Vivino : “Gonna Be 2 Of Those Days” (Gulf Coast) • Annie & The Caldwells : “Can’t Loose My (Soul)” (Luaka Bop)  • Ben l’Oncle Soul : “Sad Generation” (Enchanté) • Greentea Peng : “Tell Dem It’s Sunny” (AWAL) • Kid Ramos : “Strange Things Happening” (Nola Blue) • Kenny Wayne Shepherd & Bobby Rush : “Young Fashioned Ways” (Deep Rush • Robbert Duijf : “Silver Spoon” (Donor Productions) • Goya Gumbani : “Warworld Of The Weejuns” (Ghostly International/Modulor)  No ID : “From The Private Collection Of Saba and No ID” (From The Private Collection) Sélection parmi les sorties de ces deux dernières semaines • Emilia Sisco : “Introducing Emilia Sisco” (Timmion)  • Rhoda Scott : “Ladies And Gentlemen” (Sunset)  • The Nico Wayne Toussaint Band : “From Clarksdale With Love” (Autopublié) • Smino : “Maybe In Nirvana” (Zero Fatigue) • An Diaz & Yokatta Brothers : “Komorebi In New Orleans” (Autopublié)  • “The Soul And Songs Of Young Curtis Mayfield: The Spirit Of Chicago” (Craft) • Rashad The Blues Kid : “Live In Clarksdale” (Side2Side)  • Keziah Jones : “Alive & Kicking” (Because Music)  • Matt “The Rattlesnake” Lesch : “Blues Cut Like Glass” (Blue Lotus) • The First Cosmopolitans All Male Chorus : “Roll Jordan Roll” (Music Maker) • Parchman Prison Prayer : “Another Mississippi Morning” (Glitterbeat) Paula Breves spoke to ANBA about her experience participating in the Jameel Rally on Saudi soil earlier this year She and her teammate Vilma Rafael have been invited to another edition of the competition in 2025 and are determined to improve their ranking São Paulo – Brazilian Paula Breves, 57, has the unique experience of racing in Saudi Arabia under her belt. She and her teammate, Vilma Rafael, participated in the Rally Jameel in the Gulf country in March 2024 and are gearing up to face the competition again next year in March the race will start in Jordan but will once again pass through familiar Saudi territory “We’ve been participating in rallies for 16 years and this was our first international competition Since we are basically the only duo of Brazilian women with enough experience in this sport due to the major events we’ve taken part in we were invited by the Brazilian Automobile Confederation (CBA) to represent our country,” Breves told ANBA in an interview about her work as a driver and her experience in Saudi Arabia The Rally Jameel held its 3rd edition this year In addition to the Brazilian and Saudi participants the sporting event also featured pairs from the United States Using different equipment and vehicles than what Breves is used to the competition proved to be quite challenging “We went without our coach and had only two to three days to train with that equipment (a type of GPS) before the races started We spent nearly the entire day on each circuit I felt very honored by the invitation and gave my best to leave a good impression of our country,” says the athlete the driver shares that the competition days required a lot of adaptation from her “Although Saudi women only started driving in 2017 they’re excellent drivers and already have more experience in this type of rally which is different from what we’re used to the driver’s strength is more highly demanded navigation plays a more crucial role,” Breves recalls “I had to learn a lot in a short amount of time we finished among the top 30 in the competition and were invited to participate again in March next year then pass through locations in Saudi Arabia As she had never competed outside of Brazil before fear was also present when Breves arrived on Arab soil and we were treated well and with the utmost respect I really liked seeing how much the Arab country is encouraging women to drive and participate in rallies,” says Breves the Brazilian became a rally driver without any initial intention I started helping my husband as a navigator I’d accompany him in competitions and pay close attention to what he was doing I’ve participated in rallies in several states across the country alongside Rafael Rafael didn’t have any prior experience in the sport figuring out which type of tire we’ll have to use she found no more female competitors to race against we’re the only all-female duo competing in the Master category one of the highest levels of Brazilian rally meaning she’s responsible for taking care of the coordinates who first started participating in rallies as a hobby now says the sport has basically become her profession “When I’m not participating in rallies I work with an organization that cares for wild animals and with an app that helps organize people’s food pantry.” Breves celebrates various achievements in the sport including first place at the Mitsubishi Motorsports in 2023 and third place on the podium at the Rally Estado de São Paulo in 2015 Read more:From Brazil’s football fields to Saudi Arabia’s Report by Rebecca Vettore, in collaboration with ANBA A journalist with post-graduate degree in Digital Media from Senac, she specializes in business, economy, and entrepreneurship coverage. The Gulf country has deposited its instrument of acceptance of the World Trade Organisation (WTO) Agreement on Fisheries Subsidies, which is aimed at curbing harmful subsidies that contribute to overfishing and promoting the sustainable management of global marine resources. The Brazil-Arab News Agency (ANBA) is the news website of the Arab Brazilian Chamber of Commerce, out of São Paulo, Brazil. Its goal is to promote communication between Brazilians and Arabs. I once had a sip of a friend's breve several years ago, and it was delicious. But can you say heart-attack in a cup? I've since decided that if I were given a few months to live, I'd have one of these drinks every morning. Just because. Tell me, do you drink breves now and then? If so, how in the world do you justify them? Skip lunch and just have a breve? After all, they pack about 550 calories. is “also known as … a Brazilian aardvark,” Breves wrote He did not cite a source for this nickname He and his brother had spotted several coatis while on a trip to the Iguaçu Falls where they had mistaken them for actual aardvarks “I don’t necessarily like being wrong about things,” Breves told me I slipped in the ‘also known as the Brazilian aardvark’ and then forgot about it for awhile.” Adding a private gag to a public Wikipedia page is the kind of minor vandalism that regularly takes place on the crowdsourced Web site he assumed that someone would catch the lack of citation and flag his edit for removal Some of the most well-known examples involve Wikipedia entries for famous people such as when users edited the article on the British actor Sacha Baron Cohen to say he had worked at Goldman Sachs When a Wikipedia editor tried to remove the apocryphal detail Because it had since appeared in several articles on Cohen in the British press the burden was on Wikipedians to disprove the myth it frustrates me when journalists don’t fact check Wikipedia and end up reproducing errors because Wikipedia can only work the way it does if we have reliable sources to cite,” Stuart Geiger When theoretically trustworthy sources err who has researched the dissemination of information on Wikipedia points to the case of the Wikipedia founder Jimmy Wales’s birthday Encyclopedia Britannica said that it was August 7th Wales says that his marriage certificate contains an error and that his actual birthday is August 8th But Wikipedia and several other mainstream sources have followed Encyclopedia Britannica’s lead and listed his birthday as August 7th Though Wales has told journalists this story Wikipedia’s rules value a multitude of independent sources over the word of an article’s subject the founder of Wikipedia could not get the Web site to reflect what is—according to Wales (“Jimmy could be making this all up to make a point about Wikipedia The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission Journal of Nutritional BiochemistryCitation Excerpt :Further it's well known that probiotic strains beneficial to intestinal inflammation likely depend on their antioxidant capacity in modulating targeted miRNA expression which acts as a critical player in the maintenance of gut immune homeostasis [13] that appears compromised in intestinal inflammation [7] Previous literature that represented Bifidobacterium bifidum ATCC 29521 antioxidant role [36] and various adjacent Bifidobacterium bifidum strains strong probiotics role in different ways [37–39] convinced us to investigate this specific strain in DSS mouse model; which was not previously investigated Our study aimed to investigate the intestinal probiotic strain-specific properties of Bifidobacterium bifidum ATCC 29521 in a dextran sulphate sodium (DSS) model of mouse colitis All content on this site: Copyright © 2025 Elsevier B.V. This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Feature Films Database Southern Mediterranean films database Scriptwriters European Film Schools Production Companies Distributors International Sales Submit a Film Industry Reports Co-Production Podcast Online Screenwriting Training Course Guided Course for Feature Film Writing Script Analysis Analysis of the potential of your series Cineuropa's Training Catalogue Film Festival Photographs Newsletter Photogalleries EUFCN Location Award Euro Film Fest 27 Times Cinema GoCritic! Advertise on Cineuropa Logos and Banners PRODUCTION / FUNDING Italy by Vittoria Scarpa the directorial debut from the renowned Italian screenwriter stars Pilar Fogliati the Roman screenwriter shot the movie in Rome and Naples over the course of seven weeks Breve storia d’amore follows Lea and Rocco who meet in a bar and soon become lovers takes a sinister turn when Lea tries to wind her way into Rocco’s everyday life always with great confidence and satisfaction" "When it came to this story about twisted relationships and placing my trust in a cast of extraordinary and generous actors as well as in a crew who have been brilliant Breve storia d’amore is produced by Indigo Film, HT Film and RAI Cinema Please subscribe to our newsletter to receive more stories like this directly in your inbox 02/05/2025Production / Funding – Italy Shooting begins on Walter Fasano’s Nino, a portrait of scoring maestro Nino Rota 02/05/2025Production / Funding – Belgium Wallimage is backing Michaël R Roskam's Le Faux Soir 30/04/2025Production / Funding – Italy The final clapperboard slams on Il falsario, starring Pietro Castellitto 30/04/2025Production / Funding – UK/France/Germany Sally Potter’s Alma to star Pamela Anderson and Dakota Fanning 29/04/2025Production / Funding – Spain Claudia Pinto finishes filming Morir no siempre sale bien 29/04/2025Production / Funding – Latvia The National Film Centre of Latvia unveils the recipients of its latest round of funding Subscribe to our newsletter to receive the most important daily or weekly news on European cinema Crossing Europe 2025 Awards The New Year That Never Came and The Flats crowned at Crossing Europe Cannes 2025 Marché du Film Be For Films to sell Love Me Tender in Cannes Cannes 2025/Sponsored Latvia set to shine bright at Cannes, led by Sergei Loznitsa’s competition entry Two Prosecutors Las Palmas 2025 MECAS/Awards Manuel Muñoz Rivas and Joana Carro win awards at the eighth MECAS Cannes 2025 Marché du Film Playtime to present some high-impact and entrancing trump cards at Cannes Production / Funding Italy goEast 2025 Review: My Magical World Production / Funding Belgium Box Office Slovakia Slovak crime-thriller Černák becomes the highest-grossing film in domestic cinemas Production / Funding Italy Films / Reviews Italy Review: San Damiano Cannes 2025 16 works to be presented in the Immersive Selection at Cannes Market TrendsFOCUSA busy spring festival season awaits the European film industry. Cineuropa will continue to keep its readers up to date with the latest news and market insights, covering the buzziest events, including Cannes, Kraków, Karlovy Vary, Tribeca, Hot Docs, Annecy, Brussels, Munich and many others Distribution, Exhibition and Streaming – 02/05/2025Slovak crime-thriller Černák becomes the highest-grossing film in domestic cinemasThe second film in the saga about a local mafia boss, directed by Jakub Króner, outgrossed its first part, which dominated Slovak cinemas last year Animation – 30/04/2025Mirko Goran Marijanac • Media sales executive, DeAPlaneta EntertainmentDuring our chat, the exec shared key insights from this year’s Cartoon Next and touched on the current climate for the animation sector Želimir Žilnik • Director of Eighty Plus The Serbian director discusses his deep suspicion of ideologies in relation to his irresistibly charming latest feature, which follows a man whose life spans three political systems Paulina Jaroszewicz • Distribution and marketing manager, New Horizons Association Cineuropa sat down with the Polish distributor to discuss her company’s strategy as well as the connection between its distribution line-up and BNP Paribas New Horizons Festival’s programme Lorcan Finnegan • Director of The Surfer The Irish filmmaker discusses his mystery-thriller, how he created the character with Nicolas Cage and his approach to the use of colours in the film Julien Rejl • General Delegate, Directors’ Fortnight The General Delegate of the Cannes Directors’ Fortnight discusses the 2025 selection and clarifies the debate on its editorial line Privacy Policy The images used on this website have been provided by journalists and are believed to be free of rights if you are the owner of an image used on this website and believe that its use infringes on your copyright We will remove the image in question as soon as possible We have made reasonable efforts to ensure that all images used on this website are used legally and in accordance with copyright laws About us | Contact us | Logos and Banners Mission | Partners | Team | Donations | Terms and conditions Blame the “fukú”—a curse that has haunted Oscar’s family for generations following them on their epic journey from Santo Domingo to the USA “La Breve y Maravillosa Vida de Oscar Wao” explores the endless human capacity to persevere—and risk it all—in the name of love Know before you goThis production is performed in Spanish with English supertitles.Run time2hr VenueRepertorio Español / Spanish Theatre Repertory CategoriesPlays, Comedy, Off Broadway, Adventure, Drama Find out more information and get tickets to The Brief Wondrous Life of Oscar Wao off Broadway The running time of La Breve y Maravillosa Vida de Oscar Wao is 2hr La Breve y Maravillosa Vida de Oscar Wao is playing at Repertorio Español / Spanish Theatre Repertory The theatre is located at 138 East 27th Street Tickets for La Breve y Maravillosa Vida de Oscar Wao start at $40 Book tickets for La Breve y Maravillosa Vida de Oscar Wao on New York Theatre Guide Official TicketsDirect from the venue box office.Full Cancellation GuaranteeGet a full refund if your show is canceled.Subscribe to our newsletter to unlock exclusive New York theatre updates You can unsubscribe at any time. Privacy Policy Metrics details It has previously been shown that the consumption of probiotics may have beneficial effects not only on peripheral tissues but also on the central nervous system and behavior via the microbiota–gut–brain axis raising the possibility that treatment with probiotics could be an effective therapeutic strategy for managing neurodegenerative disorders we investigated the effects of oral administration of Bifidobacterium breve strain A1 (B breve A1) on behavior and physiological processes in Alzheimer’s disease (AD) model mice breve A1 to AD mice reversed the impairment of alternation behavior in a Y maze test and the reduced latency time in a passive avoidance test indicating that it prevented cognitive dysfunction We also demonstrated that non-viable components of the bacterium or its metabolite acetate partially ameliorated the cognitive decline observed in AD mice Gene profiling analysis revealed that the consumption of B breve A1 suppressed the hippocampal expressions of inflammation and immune-reactive genes that are induced by amyloid-β breve A1 has therapeutic potential for preventing cognitive impairment in AD Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disease that results in gradual cognitive impairment and eventually leads to dementia despite AD being one of the most prevalent neurodegenerative diseases in aging societies no clinically successful therapeutic strategies for its treatment or prevention have been reported to date it is possible that some probiotics could enable the effective therapeutic management of neurodegenerative disorders we investigated the effect of Bifidobacterium breve strain A1 (B breve A1) on the behaviors and physiological processes of AD model mice We found that this probiotic can prevent the cognitive dysfunction induced by Aβ indicating its therapeutic potential in AD patients Effect of Bifidobacterium breve strain A1 treatment on cognitive function in AD model mice evaluated by Y maze test and passive avoidance test (a) Experimental design of the mouse study An animal model of AD was induced by intracerebroventricular (ICV) injection of Aβ25–35 or Aβ1–42 The probiotics was orally administered every day starting 2 days before ICV injection cognitive function was evaluated by Y maze test thereafter the mice received passive avoidance test (d) Alternative ratio and (e) Total entry time in Y maze test of Aβ1–42 injected mice breve A1 could ameliorate memory dysfunction in mice administered Aβ Change of gene expression profile in hippocampus of AD model mice by Bifidobacterium breve strain A1 treatment using RNA-seq analysis Transcriptional analysis was performed on hippocampal tissues of sham-operated mice (SH) Aβ25-35 injected mice (AB) and mice treated with Aβ and B breve A1 (ABA) (a,b) Venn diagram of shared and unique hippocampal transcripts (a) in SH vs AB and/or SH vs ABA (c–e) GO Term enrichment analysis of differential expressed (DE) genes in AD hippocampus Enrichment analysis of differential expressed genes (c) between Aβ-treated and control mice breve A1 administration and (e) using DAVID analysis breve A1 could modulate excessive immune response induced by Aβ injection leading to ameliorating effect of Aβ toxicity Furthermore, we investigated whether administration of B. breve A1 could affect gene expression of hippocampus in sham-operated mouse. RNA-seq analysis revealed that only one gene was found to be significantly modulated in sham-operated mouse in response to administration of B. breve A1 (SHA) (Supplemental Table 2) suggesting that almost all gene expression in hippocampus was not influenced by administration of B (a,b) Plasma SCFA levels of AD model mice for acetate (a) and for propionate and butylate (b) Effect of acetate treatment and non-viable Bifidobacterium breve A1 treatment on cognitive function in AD model mice Here we showed that oral administration of B breve A1 prevented cognitive decline in AD model mice with a reduction in the immune response and neuronal inflammation breve A1 has ameliorative effects on cognitive dysfunction in both working memory and long-term memory in Aβ-injected mice was upregulated to normal level by the administration of B breve A1 prevented cognitive decline in AD model mice through its modulating effect on the immune response and neuronal inflammation implying that some structural components of the probiotics may modulate the neuronal immune response via vagus nerve stimulation further research is needed to explore whether B breve A1 directly stimulates the vagus nerve and to clarify the links between probiotics indicating that the protective effects of B breve A1 may partly be mediated by the enhanced production of acetate The mechanism of how acetate ameliorate memory dysfunction in AD mice is one of the important issues to be addressed in future study additional investigations to clarify the preventive effect of B breve A1 using tauopathy model mouse are also the important issues to be addressed in future studies the present study demonstrated that oral administration of B breve A1 to AD model mice not only improved cognitive dysfunction but also suppressed the expression of inflammation and immune-reactive genes induced by Aβ These results suggest therapeutic potential of B breve A1 for preventing cognitive impairment in AD All procedures were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals The experimental protocol was reviewed and approved by the Animal Studies Committee of Nihon Bioresearch Inc and the Animal Research Committee of Morinaga Milk Industry Co. Japan) were housed in a room with controlled lighting (12 h light/12 h dark) and a constant temperature (25 °C) and provided with MF diet (Oriental Yeast Co. breve A1 were orally administered to the mice daily by gavaging 1 × 109 organisms in 0.2 ml mice were administered sodium acetate (150 mM) in drinking water from 2 days before Aβ injection (acetate group) mice were orally administered donepezil hydrochloride (0.5 mg−1 kg−1 day−1; Wako Chemicals No adverse effects were observed following administration of any of the sample solutions each mouse was anesthetized by intraperitoneal injection of Nembutal in saline and subcutaneous injection of levobupivacaine and placed in a stereotaxic frame (Narishige Inc. 28-gauge needle was inserted to following position: 1mm right of the midline 0.2 mm posterior and 2.5 mm depth from bregma 6 nmol) was then injected intracerebroventricularly at a rate of 1 μl/min using a syringe pump The needle was kept in place for additional 3 minutes and then withdrawn Aβ protein 1–42 (Peptide Institute, Osaka, Japan) was injected into other mice at 200 pmol in 3 μl distilled water, while 3 μl distilled water was injected into a sham-operated group. Aβ 1–42 solution used in this study contained mixture of monomeric and oligomer form of Aβ (Supplemental Fig. 4) A Y maze test was performed 6 days after ICV injection to assess the working memory of the mice The maze consisted of polyvinyl plastic and had three arms (395 mm deep Mice were placed at the end of one arm and allowed to move freely for 7 min The sequence of arm entry was counted manually to calculate the total number of entries and the alternation ratio (ratio of actual alternations to maximum alternations This test was performed by a person blind to the group assignment the long-term memory of mice was evaluated by a passive avoidance test The apparatus consisted of one illuminated (100 mm wide each mouse was placed in the illuminated chamber the guillotine door was opened after 10 s and the initial latency to enter the dark compartment was recorded When the mice had moved completely into the dark compartment the door was closed and the mice received an electric shock (0.2 mA They were then returned to their home cage The test trial was conducted 24 h later by placing the mice in the illuminated chamber and measuring the latency period to enter the dark compartment up to 300 s mice were euthanized by isoflurane overdose frozen in liquid nitrogen and stored at −80 °C until use Blood was collected into a tube containing EDTA and centrifuged at 2150 × g at 4 °C for 15 min Subsequently the plasma sample was frozen in liquid nitrogen and stored at −80 °C until analysis The cecum was also removed and its contents were stored at −80 °C until use Total RNA was extracted from the hippocampi with the RNeasy® Plus Universal Mini Kit (Qiagen,Venlo Netherlands) according to the manufacturer’s instructions Poly(A)-selected RNA-seq libraries were generated using the TruSeq RNA Sample Prep Kit V2 (Illumina CA) and 150-bp paired-end sequencing was performed at BGI JAPAN Co >5% unknown bases and low quality (i.e. >20% bases with quality <15) were filtered The detailed description of the methods is provided in Supplementary Information The behavior and physiological responses of treatment groups were compared using one-way analysis of variance followed by Student’s t or Mann–Whitney U post hoc tests in PASW Statistics for Windows version 17 (SPSS Japan) Statistical analysis for RNA-seq was described in section “RNA sequencing (RNA-seq) analysis” The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request Download references We thank Hiroyasu Murasawa and Azusa Tanaka for special support for animal experiments Kanagawa Institute of Industrial Science and Technology Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences Conceptualization and methodology of the experiments: Y.K. are the employee of Morinaga Milk Industry Co. Download citation DOI: https://doi.org/10.1038/s41598-017-13368-2 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: Microbiology newsletter — what matters in microbiology research Metrics details Bifidobacteria are common gut commensals with purported health-promoting effects This has encouraged scientific research into bifidobacteria though recalcitrance to genetic manipulation and scarcity of molecular tools has hampered our knowledge on the precise molecular determinants of their health-promoting attributes and gut adaptation To overcome this problem and facilitate functional genomic analyses in bifidobacteria we created a large Tn5 transposon mutant library of the commensal Bifidobacterium breve UCC2003 that was further characterized by means of a Transposon Directed Insertion Sequencing (TraDIS) approach Statistical analysis of transposon insertion distribution revealed a set of 453 genes that are essential for or markedly contribute to growth of this strain under laboratory conditions These essential genes encode functions involved in the so-called bifid-shunt most enzymes related to nucleotide biosynthesis and a range of housekeeping functions breve core genomes highlights a high degree of conservation of essential genes at the species and genus level while comparison to essential gene datasets from other gut bacteria identified essential genes that appear specific to bifidobacteria This work establishes a useful molecular tool for scientific discovery of bifidobacteria and identifies targets for further studies aimed at characterizing essential functions not previously examined in bifidobacteria The scarcity of molecular tools for bifidobacteria has hampered progress in our understanding of the specific molecular mechanisms behind their adaptation to the intestinal ecosystem interaction with their host and possible health-promoting activities opening avenues to interrogate the functionality of bifidobacterial genes at genome level The aim of the current study was to further improve the applicability of the transposon mutagenesis method previously developed by our research group for B by applying a genome-wide random mutagenesis approach coupled to transposon mapping by means of next generation sequencing Analysis of transposon insertion distribution across the genome facilitated the identification of genes (designated here as essential genes) required for strain survival and normal growth on rich media under laboratory conditions Comparative genomic analysis between the predicted essential genes and the Bifidobacterium core genome revealed a high level of conservation of essential genes across the genus Bifidobacterium and B highlighting a set of highly conserved core metabolic functions as well as a set of still uncharacterized essential functions in bifidobacteria and allowed the identification of ~46,000 unique insertions with 1 estimated insertion in every 52 bp approximately 36,000 were mapped within the deduced coding regions of predicted ORFs Reconstruction of essential metabolic pathways and functions based on TraDIS-predicted essential genes in B Gene names or locus tags are represented in red and metabolic intermediates in grey Dashed arrows summarize multiple reactions Panels A and B represent the essential central carbohydrate utilization pathways Panel C represents essential steps within amino acid metabolism and biosynthesis pathways Panel D highlights essential steps mapped onto nucleotide metabolic pathways although they have mainly been studied in pathogenic bacteria Also two genes encoding predicted orphan ATP binding proteins (Bbr_1490 and Bbr_1890) not located in proximity to any ABC transporter-encoding genes were found to be essential in our analysis therefore suggesting they might energize crucial transport systems in the cell 53 essential genes found in this work encode hypothetical proteins with unknown functions highlighting the limited knowledge we have on associated fundamental aspects of bifidobacterial metabolism and physiology These findings also highlight the need for research on essential hypothetical proteins in order to define their biological function 11 out of the identified 453 essential genes in B breve UCC2003 correspond to transcriptional regulators that have not been functionally characterized in bifidobacteria of which nine are predicted to be involved in signal sensing and transduction therefore representing interesting targets for future studies (a) Venn Diagram representing the overlap between the essential genes determined by TraDIS analysis for B (b) Heatmap representing in colour gradient the frequency of COG categories across all the different type of gene families resulting from comparative analysis of B breve UCC2003 total gene content and TraDIS predicted essential genes against the available Bifidobacterium genomes they have not been thoroughly characterized in either B they are typically associated with mobile genetic elements the antitoxin component of a presumptive toxin-antitoxin system (encoded by Bbr_1066 and Bbr_1067) therefore suggesting the corresponding toxin component is active another chromosomal region (located between chromosome positions 907740 and 907450) which had escaped previous ORF detection and annotation efforts and displaying homology to a toxin-antitoxin module harbours uneven transposon insertions pointing towards the existence of a second active toxin-antitoxin system in the chromosome of this strain further research is required to discern the genes that are required for successful gut colonization of B breve UCC2003 (as opposed to being essential for growth under laboratory conditions) (a) Venn Diagram representing the overlap of essential genes determined by transposon sequencing in B Bacteroides fragilis 638 R and Bacteroides thetaiotaomicron VPI-5482 (b) Distribution per COG functional classification of essential genes in the three compared taxa While iron is an essential cofactor for all living bacteria specially anaerobes the fact that orthologous genes do not appear essential in Bacteroides despite their key function in the maintenance of cell homeostasis might either indicate that Bacteroides encode additional genes that can compensate the absence of one of the genes or that this systems performs other still undefined essential functions in B These findings and the high proportion of essential hypothetical functions in bifidobacteria emphasize the importance of studying genes encoding hypothetical proteins particularly because the conclusions of many (microbiome) studies heavily rely on functional predictions We identified the genes essential for growth of a commensal Bifidobacterium strain under in vitro growth conditions based on a random mutagenesis approach coupled to simultaneous mapping of the disruption point for ~46,000 different insertion sites breve core-genomes revealed significant overlap with our identified candidate essential gene set Essential genes not shared with the Bifidobacterium core genome may either represent strain-specific adaptive traits or functions required under specific environmental conditions such as those mimicking food or intestinal environments comparison between essential gene sets in other gut commensals breve UCC2003-specific essential functions which upon further study will contribute to a better comprehension of bifidobacterial physiology and evolution The current work provides a fundamental framework to gain functional insights into bifidobacterial molecular traits at a genome-wide level Expanding identification and tracking of disrupted genes in collections of bifidobacterial mutants under specific environmental conditions will assist functional annotation in this genus and contribute to a better definition of the minimal bifidobacterial genome Such knowledge in turn will help to depict a more accurate definition of the bifidobacterial lineage this work allowed identifying a set of essential functions not previously characterized in bifidobacteria including a range of hypothetical and secreted proteins the EPS cluster 1 and several orphan ATPases which represent interesting targets for further studies This PCR product was digested with XbaI and SphI and subsequently ligated to similarly digested pMOD2 (Epicentre Biotechnology) to yield pMOD2-tetT7 Transformation mixes were spread plated onto RCA agar supplemented with 0.5% ribose and 10 μg ml−1 tetracycline and plates were incubated for 2–3 days anaerobically at 37 °C plates were flooded with 1 ml of RCM supplemented with 0.5% ribose and clones were scraped from the plates using disposable sterile spreaders These bacterial suspensions were stored at −80 °C A master stock was created by pooling the mutants originated from multiple independent experiments Members of the core-genome were selected based on their presence in all strains and a filter to distinguish orthologues (single-copy) from paralogues (multiple copy) was applied Members of the core-genome were defined as genes present in each B For the comparative analyses extended at genus level breve UCC2003 was compared with draft genome sequences we decided to apply an extended core-genome that included genes present in at least 80% of the sequences (in order to exclude the possibility that genes not sequenced were considered absent) The information obtained by this clustering was employed to determine the overlap between the essential genes in each TraDIS library and visualized in a Venn diagram and heatmap The same BLAST-based approach was used to retrieve the COG classification of the essential genes described in other bacteria (belonging to the phylum Bacteroides) to be compared with B The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries Genomics of the genus Bifidobacteirum reveals species-specific adaptation to the glycan-rich gut environment Applied and Environmental Microbiology pii: AEM.03500–15 (2015) Download references LR and MOCM are members of The APC Microbiome Institute which is a research centre funded by Science Foundation Ireland (SFI) through the Irish Government’s National Development Plan The authors and their work were supported by SFI (Grant SFI/12/RC/2273) and HRB (Grant No Sequencing was supported by the Wellcome Trust CJB and AKC were supported by the Medical Research Council Lorena Ruiz and Francesca Bottacini contributed equally to this work School of Microbiology and APC Microbiome Institute Mary O’Connell-Motherway & Douwe van Sinderen created the figures and drafted the manuscript All authors read and approved the manuscript The authors declare that they have no competing interests Download citation DOI: https://doi.org/10.1038/s41598-017-05795-y Metrics details Probiotic supplementation can help to mitigate the pathogenesis of irritable bowel syndrome (IBS) by reinforcing the intestinal barrier and reducing both inflammation and proteolytic activity a combination of in vitro tests was performed on 33 Bifidobacterium strains as probiotic candidates for IBS In addition to the classical tests performed the detection of the serine protease inhibitor (serpin) enzyme capable of decreasing the high proteolytic activity found in IBS patients was included Three serpin-positive strains were selected: Bifidobacterium breve CNCM I-5644 longum CNCM I-5646 for their immunomodulation properties and protection of intestinal epithelial integrity in vitro breve CNCM I-5644 strain prevented intestinal hyperpermeability by upregulating Cingulin and Tight Junction Protein 1 mRNA levels and reducing pro-inflammatory markers The ability of CNCM I-5644 strain to restore intestinal hyperpermeability (FITC-dextran) was shown in the murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS) This effect of this strain was corroborated in a second model of IBS the neonatal maternal separation model in mice breve CNCM I-5644 may partially prevent disorders associated with increased barrier permeability such as IBS Effect of Bifidobacterium strains on IL-8 production by TNF-α stimulated HT-29 cells IL-8 production was stimulated by incubating HT-29 cells with 5 ng of TNF-α and co-incubation with Bifidobacterium strains for 6 h Comparison between treated groups and the PBS control group by a one-way ANOVA test followed by the Dunnet’s test *p < 0.05 compared to PBS control group Immunomodulatory properties of probiotic strains on human PBMCs from five donors (a) IL-10/IL-12 ratio after incubation of strains with PBMCs (b) IL-10 immunomodulation after incubation of strains with PBMCs Cytokines were quantified by ELISA after co-incubation of bacteria and PBMCs from five donors for 48 h A one-way ANOVA test was performed followed by a Dunnet’s test to compare treated groups with RPMI medium group Protective effect of Bifidobacterium serpin-positive strains on intestinal barrier integrity measured by Trans-Epithelial Electrical Resistance (TEER) TEER was measured before adding the tested strain in the apical surface of Caco-2 cells for 3 h prior to treatment of the basolateral medium with TNF-⍺ 100 ng/mL for 21 h at 37 °C *Indicates significant differences between treated groups and DMEM + TNF-α control group Serpin mRNA levels were quantified in three Bifidobacterium strains The 2−ΔΔCTmethod was carried out to quantify relative gene expression The lactate dehydrogenase gene was used as a housekeeping gene longum NCC2705 as a serpin-positive reference bacteria and B Effect of Bifidobacterium strains in the DNBS low-grade inflammation murine model (d) Fecal Lipocalin-2 (Lcn-2) and (e) Proteolytic activity Results of with a one-way ANOVA test followed by the Dunnet’s test comparing the DNBS-PBS group to others groups Impact of Bifidobacterium strains on systemic T-helper balance Cytokines secreted by splenocytes from each group of mice Results of non-parametric Mann–Whitney test comparing the DNBS-PBS group to others groups Regulation of intestinal tight junction proteins in the DNBS low-grade inflammation model (a) Intestinal permeability measured by FITC-dextran The Rpl19 and Tbp genes was used as a housekeeping gene Results of a one-way ANOVA test followed by the Dunnet’ test comparing the DNBS-PBS group to others groups breve CNCM I-5644 in the non-inflammatory NMS murine model measured as the area under the curve (AUC) of intracolonic pressure variation (IPV) was calculated by trapeze method between 60 and 100 mmHg for each group of mice (b) Intestinal permeability measured by FITC (c) and Lipocalin-2 and (d) proteolytic activity in feces Comparisons were performed by a one-way ANOVA test followed by the Dunnett’s test Results are presented as mean +/− SEM (n = 14) expressed the serine protease inhibitor “serpin” at similar expression levels to that observed with the reference strain B confirming the interest of using serpin as a criterion for probiotic selection for IBS this study highlights a wide range of immunomodulatory properties and beneficial effects on the host intestinal barrier function of several Bifidobacterium strains and provides further evidence of their efficacy to alleviate and protect the intestinal barrier in two models of IBS may be effective in preventing disorders associated with increased barrier permeability such as IBS It could be interesting to evaluate the synergistic interaction of B breve CNCM I-5644 with other probiotic strains to improve colonic hypersensitivity This work also demonstrates the importance of selecting the proper in vitro experiments such as readouts used in this study: IL-8 and IL-10 immunomodulation intestinal barrier protection and serpin detection three important probiotic characteristics for the selection of new candidate probiotic strains for the restoration of intestinal homeostasis in the context of IBS bacteria were collected 1 h after the beginning of the stationary phase re-suspended in phosphate-buffered saline (PBS; Gibco and stored at − 80 °C until further assays Colony forming units (CFU) were estimated by serial dilution on agar plates Three independent growth experiments were performed for each strain Preparation of bacterial inoculum for administration in mice was performed as described above and lyophilized cell supernatants were collected and frozen at − 80 °C until subsequent analysis of IL-8 concentrations by enzyme-linked immunosorbent assay (ELISA) (Biolegend CA) according to the manufacturer’s instructions Bacteria from three independent cultures were added at a MOI of 1:10 in 50 μL of PBS Plates were incubated for 48 h at 37 °C in 5% CO2 Samples were finally stored at − 80 °C until further analysis of IL-10 and IL-12p70 concentrations by ELISA-Multiplex (Biorad The resulting data presented as a TEER ratio: Bacterial DNA extractions were performed according to the procedures of the Instagene kit (BIORAD longum serpin and its derivatives were obtained targeting the BL0108 serpin gene insertion sequence (Genback number accession: AAN23973.1) Each PCR was performed in a thermocycler (ThermoFisher France) with the following program: initial denaturation step of 3 min at 95 °C followed by 35 cycles of denaturation for 30 s at 95 °C and extension for 1 min at 72 °C and then elongation for 5 min at 72 °C The detection was confirmed using agarose gel electrophoresis Specific pathogen-free male C57BL/6 mice (6–8 weeks old) (Janvier France) were maintained under normal breeding conditions in the animal care facilities of Infectiologie Expérimentale des Rongeurs et des Poissons (IERP) of Institut National de Recherche pour l’Agriculture Our experiments were performed in accordance with European Union legislation on animal welfare and were approved by Comité d’Éthique en expérimentation animale (COMETHEA) our local committee on animal experimentation (n°16744-201807061805486 v2) and in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) relevant guidelines All results were expressed as means ± standard error of the mean (SEM) We performed a one-way ANOVA for normal samples and multiple comparisons were carried out using Tukey’s test For non-normal samples and/or with unequal variances non-parametric tests were performed within groups (Kruskal–Wallis test) and multiple comparisons were carried out using Dunn’s test using GraphPad Prism 7 software (GraphPad Software The data generated during this study are available from the corresponding author on reasonable request Lacy, B. 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Sci. 76, 1119–1127. https://doi.org/10.1292/jvms.14-0060 (2014) Download references We would like to thank Claude Blondeau for his help for the writing assistance We wish to thank the staff of the INRAE Infectiology of Fishes and Rodents Facility (IERP-UE907 France) in which animal experiments have been performed IERP Facility belongs to the National Distributed Research Infrastructure for the Control of Animal and Zoonotic Emerging Infectious Diseases through In vivo Investigation The reported study was supported by the National Research Institute for Agriculture Paris University and a contract with PiLeJe Laboratoire Frédéric Barbut & Anne-Judith Waligora-Dupriet All read and approved the final manuscript during the conduct of this study; the other authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-022-21746-8 a leading Japanese dairy product company and a key global probiotics manufacturer confirmed that its proprietary probiotic strain Bifidobacterium breve A1 (a.k.a breve MCC1274) is safe and effective for improving memory functions of older adults with suspected mild cognitive impairment (MCI) in a randomized published recently in the Journal of Alzheimer’s Disease has produced breakthrough results uncovering a novel promising probiotic intervention for early dementia prevention The clinical study conducted by a clinical research organization in collaboration with Morinaga Milk Industry Co. is the first RCT report to show a profound cognitive enhancement benefit of the probiotic B Our findings represent an important breakthrough for MCI and dementia prevention It marks major progress in our investigation on the ability of the probiotic strain B breve A1 to halt the cognitive decline of older adults.” Director of Next Generation Science Institute of Morinaga Milk Globally, the number of people living with dementia, including Alzheimer’s disease, will increase from 50 million in 2018 to 152 million in 2050, reaching epidemic proportions. According to the Alzheimer’s Association website, it is estimated that one new patient is diagnosed with Alzheimer’s disease every 65 seconds (https://www.alz.org/alzheimers-dementia/facts-figures) causing an unprecedented burden to society MCI is a common problem in older adults associated with the risk of developing sporadic Alzheimer’s disease or other dementia within a few years if left untreated no medications have proven effective for MCI Although several trials of pharmacological treatments were thought to prevent the symptoms of MCI there is an urgent need to identify effective countermeasures to MCI and dementia Previous studies by Morinaga Milk Industry Co., Ltd. have demonstrated the potential of B. breve A1 to treat Alzheimer’s disease in a pre-clinical model and found that this probiotic strain can improve specific cognitive functions in subjects with MCI in a human clinical trial. The current study conducted on 80 healthy older adults with suspected MCI further confirmed the efficacy of B breve A1 or placebo by capsule for 16 weeks Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) total score showed a significant 11.3-point improvement in those taking A1 compared to placebo in particular for domain scores of immediate memory significant improvement of cognitive function was confirmed by another test “It was exciting to see a clear and significant improvement of memory functions such as the awareness of who The 11.3-point improvement of RBANS total score seen after only 16 weeks of daily consumption of the probiotic B breve A1 in healthy older adults with MCI is remarkable.” “Our findings could signal a profound shift as to how MCI can be treated using probiotics. Future discoveries for the precise mechanisms as to how daily consumption of B. breve A1 can improve memory will open new opportunities and alternatives for not only MCI treatment but perhaps also for other CNS diseases associated with inflammation and memory impairment.” Morinaga Milk has been focusing on the study of bifidobacteria species that are naturally present in human intestines – human-residential bifidobacteria (HRB) – for over 50 years and discovered various health benefits of its HRB strains To continue exploring the potential of its HRB strains the company has focused on the gut-brain connection – a rapidly growing area of probiotics research – and conducted various research on cognitive improvement Morinaga Milk is delighted to have uncovered the beneficial effects of its proprietary probiotic B breve A1 originating from newborn infants’ intestines is the first human-residential bifidobacteria (HRB) strain that shows great promise to improve the memory of older adults with MCI and support healthy cognitive aging This breakthrough study is yet another indication of our continuous efforts to add value to our premium line of HRB probiotic strains This promising finding will open up new business opportunities and is potentially game-changing We are excited about the potential implications of B breve A1 as a novel practical approach to promote healthy aging.” General Manager of Sales and Marketing Department Morinaga Milk Posted in: Drug Trial News | Medical Condition News Cancel reply to comment discusses how he is addressing today’s medical challenges using the technology of the future Explore how the Radian ASAP mass spectrometer is being used to streamline and enhance seized drug screening Mariangela Kosmopoulou and Tharan Srikumar Follow Bruker's experts as they discuss the SCRUM approach to software development in the scientific industry you can trust me to find commercial scientific answers from News-Medical.net please log into your AZoProfile account first Registered members can chat with Azthena, request quotations, download pdf's, brochures and subscribe to our related newsletter content A few things you need to know before we start Read the full Terms & Conditions SOUTH BEND — The Euclid Quartet performs an album release concert for “Breve” at 7 p.m 31 at Northside Hall at Indiana University South Bend the 11 single-movement tracks have only their brevity in common their styles and moods are about as disparate as classical quartet music gets the pieces include an adagio and fugue by Mozart “Breve” is dedicated to the late Ernestine M a longtime donor to Indiana University South Bend where the Euclid Quartet has served as quartet-in-residence for the past 16 years For more information, visit afinat.com and euclidquartet.com. Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1155438 This article is part of the Research TopicProbiotics for Nutrition Research in Health and DiseaseView all 15 articles Examining how host cells affect metabolic behaviors of probiotics is pivotal to better understand the mechanisms underlying the probiotic efficacy in vivo studies to elucidate the interaction between probiotics and host cells we performed a comprehensive metabolome analysis of a co-culture containing Bifidobacterium breve MCC1274 and induced pluripotent stem cells (iPS)-derived small intestinal-like cells we observed a significant increase in several amino acid metabolites including indole-3-lactic acid (ILA) and phenyllactic acid (PLA) the expression of genes involved in ILA synthesis such as transaminase and tryptophan synthesis-related genes ILA production was enhanced in the presence of purines which were possibly produced by intestinal epithelial cells (IECs) These findings suggest a synergistic action of probiotics and IECs which may represent a molecular basis of host-probiotic interaction in vivo which can act as signaling molecules and affect host physiology reported a successful co-culture of colonic organoid-derived monolayers with some anaerobic bacteria While these new technologies are still in their infancy they could advance research on host–bifidobacterial interactions These reports highlight the importance of understanding the metabolic behavior of probiotics in the small intestine In this study, we used a two-chamber co-culture device to evaluate the interaction between B. breve MCC1274, which has been reported to improve cognitive function in patients with suspected mild cognitive impairment (Xiao et al., 2020; Asaoka et al., 2022) and induced pluripotent stem cells (iPS)-derived small intestinal-like cells to construct a small intestinal tract-like environment in vitro The aim of this study was to determine how a co-culture of human IECs with the probiotic B breve MCC1274 alters their metabolic behavior breve MCC1274 was obtained from the Morinaga Culture Collection (Morinaga Milk Industry United States) supplemented with 0.05% (w/v) L-cysteine hydrochloride (Kanto Chemical Japan) for 16 h at 37°C under anaerobic conditions using an Anaero Pack (Mitsubishi Gas Chemical The culture medium was centrifuged (1,000 × g and the pellet was washed with PBS (FUJIFILM Wako Pure Chemical Japan) and suspended to approximately 1.0 × 109 CFU/ml in PBS The number of colony-forming units (CFU) on transgalactosylated oligosaccharide (TOS) propionate agar (Yakult Pharmaceutical Industry Japan) was used to calculate the number of B Monolayers of human iPS-derived small intestinal epithelial-like cells were generated from FUJIFILM human iPS cell-derived Small Intestinal Epithelial-like Cells (F-hiSIECs) (FUJIFILM Wako Pure Chemical Japan) according to the manufacturer instructions with minor modifications 0.4-μm pore polyester membrane; Greiner bio-one Austria) were coated with Matrigel (Corning United States) diluted 1:30 in DMEM/F12 medium (Gibco United States) at least 1 day before F-hiSIEC seeding thawed F-hiSIECs were suspended to 1 × 106 cells/mL in seeding medium and 340 μL of the cell suspension was seeded on each Transwell insert The F-hiSIEC culture medium was changed every 2–3 days and in the last medium change before using the co-culture device antibiotics were omitted from the F-hiSIEC culture medium Transepithelial electrical resistance (TER) values measured using the Millicell-ERS system (Merck Millipore were used to evaluate monolayer maturation The device was maintained at 37°C for 24 h before starting co-culture breve MCC1274 suspension was inoculated into the apical medium the same volume of bacterial suspensions was added to conical tubes containing an equal volume of YC medium on the apical side of the device but no IECs After incubating for 24 h at 37°C the apical medium of the device and the culture medium of the conical tube were collected to calculate the number of B breve MCC1274 by counting the number of CFU on TOS propionate agar (Yakult Pharmaceutical Industry) the culture medium was centrifuged (10,000 × g 4°C) and the supernatant was stored at −20°C until use breve MCC1274 was cultured in YC medium supplemented with 200 μM each of adenosine and xanthine (FUJIFILM Wako Pure Chemical) for 24 h under anaerobic conditions and the supernatant was stored at −20°C until analysis LC–MS/MS quantification was measured using an XBridge® C18 column (4.6 mm × 150 mm United States) and the protocols mobile phase A (1 g/L ammonium acetate) and mobile phase B (methanol) were applied at a flow rate of 0.2 mL/min Gradient elution was performed by changing the percentage of mobile phase B in the following steps: (i) maintenance of 2% for 2 min (ii) increase from 2 to 35% until 5 min (iii) increase from 35 to 75% until 21 min (iv) increase from 75 to 77% until 25 min (v) increase from 77 to 99% until 30 min (vi) maintenance of 99% until 38 min (vii) decrease from 99 to 2% until 40 min and (viii) maintenance of 2% until 55 min Six samples from two independent experiments were analyzed in this assay LC–MS/MS quantification was measured using an XBridge® C8 column (4.6 mm × 150 mm United States) and the protocols mobile phase A (0.5 g/l ammonium formate) and mobile phase B (methanol) were applied at a flow rate of 0.2 ml/min (ii) increase from 2 to 65% until 40 min (iii) increase from 65 to 99% until 45 min (iv) maintenance of 99% until 55 min (vii) decrease from 99 to 2% until 60 min and (viii) maintenance of 2% until 75 min Three samples from one experiment were analyzed in this assay Functional classification and reconstruction of metabolic pathways of annotated DEGs were performed using KEGG Mapper (v 5.0) Five samples monocultured in tubes and six co-cultured samples from two independent experiments were analyzed the relative abundance of each metabolite in the metabolome analysis and the concentrations of ALAs and aromatic pyruvic acids The comparison of the effects of purines for ALAs synthesis was assessed using Dunnett’s test principal component analysis (PCA) and heatmap analyses were performed with the scaled value of the annotated peak intensity The value of no detection (N.D.) in the metabolomics analysis data was regarded asset at eps (2−52) Heatmaps described the significantly different metabolites (q-value <0.1) as calculated with the Kruskal–Wallis test and Benjamini–Hochberg post-hoc test Vertical and horizontal hierarchical clustering was calculated using Spearman’s correlation distance and Ward’s clustering method genes with a q-value <0.05 and a fold change (FC) of >2.0 were defined as DEGs Statistical analyses were performed using R (version 4.1.3) with the FactoMineR package (version 2.4) and factoextra package (version 1.0.7) for PCA analysis the ComplexHeatmap package (version 2.10.0) for heatmap analysis and the Enhancedvolcano package (version 1.14.0) for visualizing DEGs of RNA-seq data Initially, we confirmed the functionality of the device by evaluating the metabolites in the apical (YC medium) and basolateral sides (F-hiSIEC culture medium) of IECs without co-cultivation with bacteria. Based on total peak-annotated metabolites, we confirmed that distinctly different clusters were formed in the medium of the basolateral and apical sides, as can be seen in PCA (Supplementary Figure S2A) and heatmap analysis (Supplementary Figure S2B) These data indicate that there were less leaks of metabolites from one side to the other suggesting that IECs were not in a leaky state and this device is suitable for subsequent experiments Comparison of the metabolites in the culture supernatants of Bifidobacterium breve MCC1274 with or without IECs (A) PCA plot based on the total metabolites detected in the apical side of the device breve MCC1274 monocultured for 24 h in tube supernatant of IECs monocultured in the apical side of the co-culture device cultured for 48 h; IECs + Bif breve MCC1274 in the apical side of the co-culture device cultured for 24 h breve MCC1274 before (0 h) and after (24 h) cultivation in the tube as a monoculture and in the device as a co-culture with IECs The data represent the average of two samples (0 h) and six samples (24 h) from each group from two independent experiments (Average ± standard deviation) Standard deviation were not described in before cultivation samples (C) A heatmap of the scaled intensity value of each metabolite with a significant difference (q-value <0.1) in the Kruskal–Wallis and Benjamini–Hochberg tests To explore the different metabolites in each group, cluster classification based on the relative abundance of each metabolite was performed (Figure 1C; Supplementary Table S2) The metabolites were divided into six clusters as follows: the metabolites with higher abundance in the culture medium of IECs the metabolites with higher abundance in the applied medium which is subdivided into two clusters IV-1 and IV-2 the metabolites with higher abundance in the applied medium without IECs the metabolites with higher abundance in the culture medium of B These results suggest that the increase in ALAs in the co-culture conditions was attributed to metabolic changes in either B and not due to a substrate increase in the co-culture environment Figure 2. Peak intensity of amino acid-derived metabolites and their precursor amino acids. (A) Amino acid-derived metabolites, alternatives are shown in parentheses when peaks were annotated with more than one metabolite. (B) Precursor amino acids corresponding to the five metabolites shown in Figure 3 (A) Data are expressed as the mean ± SD (n = 3),* p < 0.05 ** p < 0.01 (Welch’s t-test) Since some metabolites shown in Figure 2A were not annotated as a single substance using CE-FTMS, we performed LC–MS/MS to quantify ALAs and the corresponding aromatic pyruvic acids (Figure 3) We confirmed that PLA and ILA concentrations were significantly increased (p < 0.01) while 4-OH-PLA concentration showed a tendency to increase (p = 0.09) in the co-culture conditions compared with the concentrations in the monoculture of B The concentration of the precursor metabolite phenylpyruvic acid (PpyA) significantly increased in the co-culture group (p < 0.01) while 4-hydroxyphenylpyruvic acid (HpyA) was only detected in the co-culture group the level of indole pyruvic acid (IpyA) was under detection limit (data not shown) Quantitative analysis of aromatic lactic acids and their precursor metabolites The concentrations of aromatic lactic acid-related metabolites The data represent the average of six samples from each group **p < 0.01 (Welch’s t-test) N.D. suggesting that amino acid metabolism by B breve MCC1274 increased in the co-culture with IECs Comparison of gene expression profiles of B breve MCC1274 in monoculture and in co-culture with IECs Vertical and horizontal dotted lines indicate the fold-change value |FC| = 2 and a q-value of 0.05 Upregulated DEGs in the co-culture conditions are labeled in red while downregulated DEGs are labeled in blue RNA-seq was performed on five to six samples from two independent experiments (B) KEGG Orthology (KO) functional classification of annotated DEGs The horizontal axis represents the number of genes Categories I–V represent the following mapped KEGG pathway categories: I (D) The normalized read counts from RNA-seq analysis breve MCC1274 monocultured in the tube; Co **q < 0.01 (Deseq2) (E,F) Tryptophan synthesis pathway components: (E) NADH synthesis (F) The red arrow indicates the enzymes whose gene expression was upregulated in co-culture (fold change >2 Black arrows indicate gene expression that was not significantly changed The numbers next to the boxes describing KEGG Orthology indicate fold change 2-Dehydro-3-deoxy-D-arabino-heptonate 7-phosphate (A) The purine metabolism pathway from adenine to uric acid (B) Peak intensity of purine metabolites detected using CE-FTMS **p < 0.01 (Welch’s t-test) (C) Aromatic lactic acid concentrations when cultured in the YC medium with purines **p < 0.01 (Dunnett’s test) Data are expressed as the mean ± SD (n = 3) This suggests that the intestinal barrier function was maintained even when the co-culture devise was put under the anaerobic conditions for 48 h the gene expression of aat was significantly increased and that of ldh4 showed a tendency to increase These results imply that under the co-culture conditions the expression of both lactate dehydrogenase and transaminase involved in ALAs production was upregulated which probably contribute to the conversion of aromatic amino acids into aromatic lactic acids via aromatic pyruvic acids there was an enhancement in genes involved in tryptophan and NADH synthesis Tryptophan is the substrate for ILA synthesis NADH acts as a cofactor for the dehydrogenase reaction The data from this study imply that in the co-culture not only the gene expression directly related to ALA synthesis is enhanced but so it substrate and cofactor synthesis which ultimately increased PLA and ILA concentrations The relative abundance of purines in the medium was altered in the presence of IECs. Our results suggest that the increase in ILA in the co-culture with IECs was partly due to purine metabolites derived from IECs. Since the enzymes involved in purine metabolism, converting adenosine to uric acid, were highly expressed in the human small intestine (Uhlen et al., 2010) (Human Protein Atlas proteinatlas.org) food-derived adenosine could also convert to inosine The mechanism underlying the enhancement of ILA production in the B breve MCC1274 via supplementation of hypoxanthine and xanthine is yet to be elucidated Another possible mechanism related to the enhancement of ALAs production in the co-culture is that IECs and microbes collaborate to synthesize ALAs substrates. Humans possess a gene encoding transaminase for metabolizing phenylalanine and tyrosine to PpyA and HpyA, respectively. In this study, IECs cultured alone produced the same amount of PpyA as B. breve MCC1274 cultured alone (Figure 3) a significant enhancement in PLA concentration was observed the effect of IECs on PpyA synthesis cannot be ruled out breve MCC1274 or IECs will determine the main producer of PpyA While we attempted to mimic the in vivo gut environment to provide relevant results several limitations remain to be addressed our model lacked some important factors present in vivo such as the microbiota and bile acids present in the small intestinal environment the in vitro three-dimensional structure of IECs future studies should use a system that best mimic the in vivo environment there was an enhancement in the expression of genes involved in amino acid biosynthesis the observed changes in the gene expression were not directly linked with extracellular metabolic shift in this study Investigating the intracellular metabolites in B breve MCC1274 will provide insights into this enhancement collection of an appropriate bacteria cell numbers is the most challenging issue when using this co-culture system we demonstrated that IECs could affect the metabolism of the probiotic strain B breve MCC1274 using a host-microbe co-culture system the amounts of the immunomodulatory metabolites PLA and ILA increased in the presence of IECs compared with concentrations in monoculture conditions the concentrations of aromatic amino acids were not increased in co-culture conditions Our findings revealed an enhancement in the expression of some B breve MCC1274 genes in the presence of IECs hypoxanthine and xanthine derived from IECs was linked with increased ILA production by B These results suggest that IECs-derived factors may enhance the synthesis of beneficial metabolites The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material ToK and TaK: co-cultivation device development and supervision and editing and reviewing manuscript YY and TH: co-cultivation device development All authors contributed to manuscript revision and read and approved the submitted version We thank FujiFilm Wako Corporation for their guidance in culturing F-hiSIEC, and for providing us with antibiotics-free F-hiSIEC culture medium, Takuma Sakurai for supporting LC–MS/MS analysis, Ayako Horigome for supporting RNA-seq analysis, and Editage (www.editage.com) for English language editing and TO were employed by Morinaga Milk Industry Co. The remaining 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 claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1155438/full#supplementary-material capillary electrophoresis Fourier-transform mass spectrometry; CFU Kyoto Encyclopedia of Genes and Genomes; LC–MS/MS liquid chromatography–tandem mass spectrometry; PBS Transgalactosylated oligosaccharide; 4-OH PLA The genus Bifidobacterium: from genomics to functionality of an important component of the mammalian gut microbiota Effect of probiotic Bifidobacterium breve 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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: Akira Sen, YWtpcmEtc2VuNDY4QG1vcmluYWdhbWlsay5jby5qcA== 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 Bifidobacteria are common members of the gastro-intestinal microbiota of a broad range of animal hosts Their successful adaptation to this particular niche is linked to their saccharolytic metabolism which is supported by a wide range of glycosyl hydrolases In the current study a large-scale gene-trait matching (GTM) effort was performed to explore glycan degradation capabilities in B By correlating the presence/absence of genes and associated genomic clusters with growth/no-growth patterns across a dataset of 20 Bifidobacterium breve strains and nearly 80 different potential growth substrates we not only validated the approach for a number of previously characterized carbohydrate utilization clusters but we were also able to discover novel genetic clusters linked to the metabolism of salicin and sucrose genetic associations were also established for antibiotic resistance and exopolysaccharide production thereby identifying (novel) bifidobacterial antibiotic resistance markers and showing that the GTM approach is applicable to a variety of phenotypes the GTM findings clearly expand our knowledge on members of the B in particular how their variable genetic features can be linked to specific phenotypes The current study represents a substantial expansion of our previous phenotypic investigation of this species breve genomes tested for their growth performance on 77 different saccharidic substrates we identified two previously uncharacterized carbohydrate utilization gene clusters extension of our approach to EPS production and antibiotic resistance resulted in novel findings which significantly expands our knowledge on members of this bifidobacterial species Comparative genomics of 20 Bifidobacterium breve strains Representation of comparative genomics and pan-genome analysis conducted on 20 B breve strains and additionally compared to 53 publicly available B Panel A: Heatmap representing two-way hierarchical clustering analysis (HCL) conducted on the 20 B Estimation of core-genome and dispensable-genome is also indicated in gene families (GF) Panel B: Circular plot representing the distribution of MCL families among B breve strains relative to their position along the chromosome Absence of families are indicated by white regions while elements of core-genome and dispensable-genome are also highlighted consisting of the 20 strains used in this study as well as 53 additional Pan-genome and core-genome sizes are indicated as well as the previously estimated pan-genome size (Bottacini et al. breve glycobiome and fermentation profiles Representation of the distribution of glycosyl hydrolases (GHs) across B Panel A: Predicted GH-encoding gene content of 20 B breve genomes displayed as a presence/absence heatmap An additional bar chart indicates the abundance of each GH family across the 20 B Panel B: Heatmap representing the fermentation profiles of the 20 B breve strains as based on 77 different substrates For this reason and with the aim of finding possible substrates to a relatively high and uncharacterized β-glucosidases in our dataset (15 families) available plant-derived β-glucans and natural phenols (e.g raftelin and amygdalin) were incorporated as substrates to be tested in our study (see below) GTM analysis applied to carbohydrate utilization by B. breve. GTM in B. breve conducted for 337 presence/absence clusters and 37 potential growth substrates. Substrates were excluded from the displayed analysis if they supported growth of all tested strains or if no growth was observed for any of the strains (Supplemental Table S3) Panel A: Heatmap showing gene clusters that match growth patterns on seven substrates (cellobiose Panel B: Insertional mutagenesis and assessment of the sucrose utilization in B breve UCC2003-0020/21 insertional mutants in sucrose the GTM method adopted here not only allowed us to corroborate our previous findings but also facilitated the identification of genes involved in sucrose and salicin metabolism and this NRBB52-associated gene cluster was indeed shown to be required for utilization of salicin Of the 35 substrates for which differential growth was observed among B did not return any useful positive matches when employing GTM utilization of certain carbohydrates may be influenced by factors other than the simple presence/absence of genes (e.g but also involvement of multiple distinct pathways or point mutations) and in such cases a different approach will be required to identify the genes involved GTM analysis applied to antibiotic resistance in B breve genomes with corresponding phenotypic assays Panel A: Heatmap representing presence /absence of identified antibiotic resistance markers across 20 B breve strains matching the observed phenotype Panel B: Locus map representing the surrounding regions of the identified antibiotic resistance markers (e.g erythromycin and aminoglycoside resistance) returning positive match in GTM breve 139W4-23 the tetW homolog is surrounded by multiple transposases suggesting acquisition by horizontal transfer thus resulting in the identification of several antibiotic selection markers which in turn may be used to develop novel genetic tools specific for bifidobacteria In order to extend our GTM analysis to a third phenotype we investigated whether the presence/absence of certain exopolysaccharide (EPS)-biosynthesis related genes would result in a corresponding EPS production phenotype and furthermore deduce a possible consensus for B GTM analysis applied to EPS production by B GTM applied to EPS production phenotype in B Panel A: Heatmap representing the EPS-associated genetic functions of which presence matched the observed EPS production phenotype (red) across the assessed B Five strains showing discrepant genotype/phenotype association are italicized Non-EPS-producers clearly show apparent reduction in OD as they ‘sediment’ to the bottom of the tube while EPS producers do not exhibit such a sedimentation phenotype In the case of EPS regions of a size between 30 and 50 Kb either EPS “producers” or “non-producers” were observed suggesting that EPS biosynthesis gene clusters of this size may still contain all genetic information for EPS production breve NRBB09) indeed corresponded to the expected EPS-negative phenotype our comparative and GTM analysis when applied to EPS production in B breve defined gene functions which can be used for in silico discrimination between potential EPS “producers” and “non-producers” thus facilitating the in silico screening of B Comparative genome analysis represents a powerful bioinformatics tool to assess gene distribution across members of a given species If combined with pan-genomic extrapolations gene comparisons may also allow identification of genes that are responsible for strain diversification The presence of gene families that are variably present across members of a given species can be used as a starting point for phenotypic investigations using GTM breve representatives for a number of different phenotypes generated novel information about the implications of the genetic diversity observed for this species (e.g carbohydrate utilization capabilities and EPS biosynthesis) The main advantage of GTM as compared to classical approaches generally based on single strain investigation and not employing comparative genomic analysis yet accurate method to pin-point variably distributed genes being responsible for different phenotypic traits Our findings indeed show that GTM can be used to identify genes involved in carbohydrate utilization in B breve and by extension (bifido)bacteria in general Expansion of this analysis to a different phenotypes allowed the identification of novel antibiotic resistance markers which may be useful to develop novel cloning vectors specific for B the application of GTM to a more complex phenotype such as capsule biosynthesis constituted a challenge for the analysis in particular because EPS production is the result of a combination of presence and co-occurrence of multiple genes in this case the comparative genome analysis and GTM allowed us to identify genetic functions linked to the observed phenotype thereby facilitating the distinction between EPS “producers” and “non-producers” our study shows that comparative genome analysis can be integrated with phenotypic investigations in order to identify candidate genes responsible for various phenotypic traits thereby generating new information on the unique genetic features of members of this bifidobacterial species In order to apply GTM analysis to carbon source utilization identified gene families were filtered to exclude those that are present in all strains as not contributing to strain diversification integrated episomes and prophages) and phage-defence mechanisms (e.g CRISPR/Cas and restriction-modification systems) which are not assumed to be associated with carbon source utilization The gene families obtained in this manner were further clustered into unique combinations of occurrence across strains and a binary matrix was then deduced from the dataset (values 0 for absence and 1 for presence of a cluster) The thus generated “genotype” matrix contains 337 clusters as rows and 20 strains as columns The same approach was adopted to cluster the obtained fermentation profiles resulting in a binary matrix constituting the “phenotype” For this 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Proceedings of the National Academy of Sciences of the United States of America 109, 2108–2113, https://doi.org/10.1073/pnas.1115621109 (2012) Download references This work was sponsored by Nutricia Research ME and JvB are members of The APC Microbiome Ireland The authors and their work were supported by SFI (Grant SFI/12/RC/2273) IRC Grant (GOIPD/2017/1302) and HRB Grant (PDTM/2011/9) We thank the Department of Agriculture Food and Marine (DAFM) for supporting the INFANTMET (Infant Nutrition for Programming the Gut Microbiota in Neonates) project which allowed the isolation of some of the B breve strains used in this study; we acknowledge all students and co-workers for their contribution and enthusiasm Longlife and Hayashibara for the gift of carbohydrates Denmark) for the provision of purified HMOs Mary O’Connell Motherway & Douwe van Sinderen and conceived the study together with J.L. performed all the bioinformatic and data analysis KvL and JK are employees of Nutricia Research Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-018-28919-4 2022 at McLeod Regional Medical Center of the Pee Dee son of Baltazar Rodriquez Covarrubias and Ingrid Breve-Juarez He was a 7th grader at Southside Middle School He was a star player for Youth Soccer of SC Surviving in addition to his parents are his siblings Antony Simon Juarez and Guel Rodriguez Breve Ana Rosa Rodriguez Covarrubias of Coward; grandfather with burial to follow at the church cemetery The family will receive friends from 12:00 PM – 5:00 PM prior to the funeral service at the church Metrics details breve strain UCC2003 possesses specific metabolic pathways for the utilisation of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) which represent the central moieties of Type I and Type II human milk oligosaccharides (HMOs) Using a combination of experimental approaches the enzymatic machinery involved in the metabolism of LNT and LNnT was identified and characterised Homologs of the key genetic loci involved in the utilisation of these HMO substrates were identified in B and were shown to be variably present among other members of the Bifidobacterium genus with a distinct pattern of conservation among human-associated bifidobacterial species Schematic structures of Type I HMO moiety LNT all of which feed into the overall Bifidobacteriaceae-specific metabolic pathway known as the Bifid Shunt breve possesses the metabolic machinery for the degradation and utilisation of LNT and LNnT we assess the presence and distribution of key gene loci involved in LNT and LNnT utilisation across members of the Bifidobacterium genus The genes upregulated in expression included those corresponding to the loci Bbr_0526-530 The possible involvement of these genes in LNT/LNB metabolism will be further discussed below Based on the microarray results and functional prediction of these LNT (and LNB)-upregulated genes, we implicate the gene clusters Bbr_0526-0530, Bbr_1554-1560, Bbr_1585-1590 and possibly Bbr_1551-1553 (outlined in Fig. 2) in LNT and LNB metabolism in B. breve UCC2003. Schematic representation of the gene loci involved in the utilisation of LNT The length of the arrows is proportional to the size of the open reading frame and the gene locus name which is indicative of its putative function Genes shown in red are predicted to encode proteins with an intracellular localisation genes shown in green are predicted to encode proteins with a transmembrane localisation and genes shown in blue are predicted to encode proteins with an extracellular localisation and a signal peptide sequence where the genes were designated gosR (lntR) we chose to re-designate this cluster as the lnt cluster as its primary function appears to be in LNT and LNnT metabolism (see below) which are all predicted to function in the metabolism of GNB/LNB HPAEC chromatogram profiles of (a) LNT and (b) LNnT when incubated in MOPS buffer (pH7) with: (I) LntA alone followed by a denaturation step and the subsequent addition of NahA When NahAHis was incubated alone with LNT, no degradation of the tetrasaccharide structure was observed (Fig. 3a) and NahAHis were together incubated with LNT complete breakdown of LNT to the monosaccharide constituents was observed followed by an enzymatic heat denaturation step different reaction product profiles were observed Samples taken following the initial denaturation prior to the addition of NahAHis showed the presence of lacto-N-triose and galactose Samples then taken following subsequent incubation with NahAHis indicated the presence of galactose These results show that LntA hydrolyses LNT The lactose is then further broken down by LntA (and probably other β-Galactosidases in vivo) (a) Final OD600nm values after 24 hours of growth of wild type B breve UCC2003-lacZ6 in modified MRS containing 1% (wt/vol) lactose 1% (wt/vol) LNT or 1% (wt/vol) LNnT as the sole carbon source (b) Final OD600nm values after 24 hours of growth of wild type B breve UCC2003-lnbP in modified MRS containing 1% (wt/vol) lactose 1% (wt/vol) LNnT as the sole carbon source The results are the mean values obtained manually from two separate experiments (due to the limited availability of certain carbohydrates) Error bars represent the standard deviation confirming the crucial role of lnbP in LNB metabolism while it also shows that lntA plays no direct in vivo role in LNB metabolism These findings demonstrate that nahA is crucial for LNT metabolism being responsible for the hydrolysis of lacto-N-triose The genes upregulated in expression included those located in the loci Bbr_0526-530 Possible involvement of these genes in LNnT/LacNAc metabolism are assessed below Based on the results of the microarray analyses performed and functional annotation of LNnT/LacNAc-upregulated genes, we propose that the products of the gene clusters Bbr_0526-0530, Bbr_1551-1553 and Bbr_1554-1560 (schematically outlined in Fig. 2) are involved in the metabolism of LNnT and LacNAc (present as central moieties in Type II HMO) in B demonstrating a triple specificity for Galβ-1,3GlcNAc Galβ-1,4GlcNAc and Galβ-1,4Glc glycosidic linkages and thus both Type I and Type II HMO central moieties and lactose also capable of hydrolysing LNnT and lactose (data not shown) Similar results from separate and combined reactions were obtained using LacZ2His or LacZ6His, together with NahAHis on the substrate LNnT (Supplemental Fig. S2) These results agree with the model for Type II HMO metabolism proposed here; where LntA LacZ2 and/or LacZ6 (and perhaps other β-galactosidases) hydrolyse LNnT which neither purified LacZ2 nor LacZ6 displayed the ability to hydrolyse (data not shown) Lactose is then further broken down by LntA (and other β-Galactosidases these two β-Galactosidases may carry out hydrolysis of Type II HMO central moieties (in conjunction with LntA) suggesting lnbP plays no direct role in LNnT utilisation This result demonstrates the essential role of the nahA product in LNnT metabolism by hydrolysing lacto-N-triose at its GalNacβ1-3Gal linkage breve UCC2003-lacS did reach final OD600nm values similar to that of UCC2003 when grown on ribose (data not shown) Heatmap representing the distribution of homologs of two genes from B bifidum PRL2010 across the Bifidobacterium genus Gene products from the representative strain genomes of all online-available Bifidobacterium species with a significant homology of 70% iterative similarity over 50% of protein length are represented in the matrix which employs a code colour grading that represents the degree of sequence similarity with species ordered by origin of isolation Bbr_1556 (nahA) and Bbr_1587 (lnbP) were selected from B Schematic representation of the proposed model for the metabolism of free LNT demonstrated the preferential activities of one GH42 family glycoside hydrolase (Bga42A) in hydrolysing Type I HMOs and one GH2 family glycoside hydrolase (Bga2A) in hydrolysing Type II HMOs and lactose The transcriptomic results clearly implicate nahS lntP1 and lntP2 in the utilisation on both LNT and LNnT being involved in the internalisation of these sugars into the cell The inability of the UCC2003-nahS mutant to grow on LNnT suggests the role of the nah locus-encoded transporter as the sole system responsible for LNnT internalisation by UCC2003 since UCC2003-nahS displays growth on LNT comparable to that of the wild type strain the nah transport system may not be involved in LNT transport but with its function aided by one or more additional transport systems The ability of the UCC2003-lntP1 and UCC2003-lntS to grow in mMRS supplemented with LNT demonstrates that the lnt transport system is also not exclusively We therefore suggest that either these two transport systems may have at least partially overlapping substrate specificities or that another yet undetermined transport system is partially or wholly responsible for the internalisation of LNT which explains the presence of this pathway in B which likely ‘sweeps up’ the free LNB released by the extracellular hydrolysis of larger HMO structures by other microbiota members and then utilise it via the GNB/LNB pathway all of these appear to be extracellular proteins as opposed to the (predicted) intracellular localisation of their B the biggest defining factor separating the B bifidum model appears to be the hydrolysis pathway of Type I central moiety LNT bifidum PRL2010 LnbB hydrolyses LNT extracellularly at its GlcNAcβ1-3Gal linkage and phosphorolysed by lnbP1 and lnbP2; and lactose which is hydrolysed by β-galactosidase activities the nahA homologs are expected to play a similar role in Bifidobacterium species associated with these hosts This suggests a common adaptation of the LNT/LNnT-utilisation pathway among bifidobacteria associated with the primate gut using their respective milk oligosaccharides as substrates thereby explaining co-evolution with and colonisation of this host Carbohydrates used were lactose (Sigma Aldrich lactosamine-hydrochloride (lactosamine-HCl) (Glycom A 1% wt/vol concentration of carbohydrate was considered sufficient to analyse the growth capabilities of a strain on a particular carbon source The addition of these carbohydrates did not significantly alter the pH of the medium and therefore subsequent pH adjustment was not required Growth profiles of sixteen distinct Bifidobacterium breve strains from the UCC collection (listed in Supplemental Table S2) on LNT or LNnT LNB and lactosamine-HCl were not included in these assays as sufficient (and affordable) quantities of these carbohydrate substrates could not be obtained breve UCC2003 generated in this and other studies LNnT or LNB as carbohydrate source and in each case using lactose as a positive control The correct orientation and integrity of all plasmid constructs (see also below) were verified by DNA sequencing by electrotransformation and transformants were selected based on tetracycline and chloramphenicol resistance The ligation mixtures were introduced into L lactis NZ9000 by electrotransformation and transformants were then selected based on chloramphenicol resistance The plasmid content of a number of Cm-resistant transformants was screened by restriction analysis and the integrity of positively identified clones was verified by sequencing a 50-μl volume of each purified protein (protein concentration of 0.5 mg/ml) was added to 20 mM morpholinepropanesulfonic acid (MOPS) (pH 7.0) buffer and 1 mg ml−1 (wt/vol) of one of the above-mentioned sugars in a final volume of 1 ml followed by incubation for 24 hours at 37 °C a sample was heated to 85 °C for 15 minutes following 12 hour incubation with the first enzyme and a given substrate before the addition of the addition of a second enzyme which was then followed by a further 12-hour incubation at 37 °C All samples were subject to a final enzyme denaturation step at 85 °C for 15 minutes Carbohydrate fractions from the above-mentioned hydrolysis assays (25 μl aliquots) were separated on a CarboPac PA1 analytical-exchange column (dimensions 250 mm by 4 mm) with a CarboPac PA1 guard column (dimensions 50 mm by 4 mm) and a pulsed electrochemical detector (ED40) in PAD mode (Dionex) Elution was performed at a constant flow-rate of 1.0 ml/min at 30 °C using the following eluents for the analysis: eluent A 100 mM NaOH plus 550 mM Na acetate; eluent C The following linear gradient of sodium acetate was used with 100 mM NaOH: from 0 to 50 min Chromatographic profiles of standard carbohydrates were used for comparison of the results of their breakdown by LntA Chromeleon software (version 6.70; Dionex Corporation) was used for the integration and evaluation of the chromatograms obtained A 1 mg/ml stock solution of each of the carbohydrates as well as their putative breakdown products (where available) used as reference standards was prepared by dissolving the particular sugar in Milli-Q water The resulting output was used to first build a presence/absence binary matrix and then the genes of interest were selected and represented in a heatmap employing a code colour grading that represents the degree of sequence similarity longum JCM1217 and BBPR_1438 (lnbB) was selected from B The microarray data obtained in this study have been deposited in NCBI’s Gene Expression Omnibus database and are accessible through GEO Series accession number GSE84710 Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Human milk oligosaccharides are resistant to enzymatic hydrolysis in the upper gastrointestinal tract The American Journal of Clinical Nutrition 71 OLIGOSACCHARIDES IN HUMAN MILK: Structural Human microbiota: ‘The philosophers have only interpreted the world in various ways Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis 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neutral oligosaccharides levels in human colostrum Human milk oligosaccharides and their potential benefits for the breast-fed neonate Bifidobacterial utilization of human milk oligosaccharides International Journal of Food Microbiology 149 doi: 10.1016/j.ijfoodmicro.2011.01.025 (2011) Glycoprofiling of Bifidobacterial Consumption of Human Milk Oligosaccharides Demonstrates Strain Specific Preferential Consumption of Small Chain Glycans Secreted in Early Human Lactation Journal of Agricultural and Food Chemistry 55 The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants Effect of Breast and Formula Feeding on Gut Microbiota Shaping in Newborns Frontiers in Cellular and Infection Microbiology 2 Establishment and development of lactic acid bacteria and bifidobacteria microbiota in breast-milk and the infant gut doi: 10.1016/j.anaerobe.2010.02.004 (2010) International journal of systematic and evolutionary microbiology 61 Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron Bifidobacterial enzymes involved in the metabolism of human milk oligosaccharides infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp doi: 10.1016/j.anaerobe.2012.04.012 (2012) Distribution of in vitro fermentation ability of lacto-N-biose I a major building block of human milk oligosaccharides a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217 Acta Crystallogr Sect F Struct Biol Cryst Commun 63 Cooperation of beta-galactosidase and beta-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure The genome sequence of Bifidobacterium longum subsp infantis reveals adaptations for milk utilization within the infant microbiome Proceedings of the National Academy of Sciences of the United States of America 105 Intra- and Extracellular β-Galactosidases from Bifidobacterium bifidum and B doi: 10.1128/AEM.67.5.2276-2283.2001 (2001) Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression Variation in consumption of human milk oligosaccharides by infant-gut associated strains of Bifidobacterium breve Metabolism of sialic acid by Bifidobacterium breve UCC2003 A key genetic factor for fucosyllactose utilization affects infant gut microbiota development Ribose utilization by the human commensal Bifidobacterium breve UCC2003 doi: 10.1111/j.1751-7915.2009.00152.x (2010) Transcriptional and functional characterization of genetic elements involved in galacto-oligosaccharide utilization by Bifidobacterium breve UCC2003 Identification of N-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway in Bifidobacterium longum Novel Putative Galactose Operon Involving Lacto-N-Biose Phosphorylase in Bifidobacterium longum doi: 10.1128/AEM.71.6.3158-3162.2005 (2005) Ability of Bifidobacterium breve To Grow on Different Types of Milk: Exploring the Metabolism of Milk through Genome Analysis Cross-feeding by Bifidobacterium breve UCC2003 during co-cultivation with Bifidobacterium bifidum PRL2010 in a mucin-based medium Beta-1,3-galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization partial purification and relation to mucin degradation Biotechnology and applied biochemistry 29 (Pt 1) A two-component regulatory system controls autoregulated serpin expression in Bifidobacterium breve UCC2003 Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003 Molecular cloning and comparative analysis of four beta-galactosidase genes from Bifidobacterium bifidum NCIMB41171 Crystallographic and mutational analyses of substrate recognition of endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum Evolutionary Glycomics: Characterization of Milk Oligosaccharides in Primates A medium for the cultivation of 688 lactobacilli Selective carbohydrate utilization by lactobacilli and bifidobacteria Improved medium for lactic streptococci and their bacteriophages Carbohydrate catabolic diversity of bifidobacteria and lactobacilli of human origin International journal of food microbiology 203 doi: 10.1016/j.ijfoodmicro.2015.03.008 (2015) Artemis: sequence visualization and 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Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Download references The authors would like to sincerely thank Glycom A/S (Lyngby Denmark) for the provision of purified HMO samples used in this study under their donation program This study was funded in part by the Irish Research Council under the Postgraduate Research Project Award; Project ID GOIPG/2013/651 the authors are supported by Science Foundation Ireland (SFI) (Grant No SFI/12/RC/2273) and Mary O’Connell Motherway is a recipient of a HRB postdoctoral fellowship (Grant No Francesca Bottacini & Douwe van Sinderen All authors analysed the results and contributed to writing the manuscript The authors declare no competing financial interests Download citation Sorry, a shareable link is not currently available for this article. 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Volume 12 - 2021 | https://doi.org/10.3389/fmicb.2021.653587 Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains we characterized EPS production by a panel of human B breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific Using wild type and isogenic EPS deficient mutants of B breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) breve UCC2003 EPS negative (EPS–) strain increased expression of cytokine genes (Tnfa and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain breve UCC2003 and JCM7017 EPS– strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80 breve UCC2003 and JCM7017 EPS– strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B This study identifies a new immunomodulatory role for B breve EPS and suggests it may be important for immune evasion of adaptive immunity by B breve and contribute to host-microbe mutualism breve EPS production was required to prevent maturation of dendritic cells and dendritic cell-mediated activation of CD4+ T cells responses Transformation of the pMG-mCherry-OVA plasmid into B breve strains was achieved by electroporation with subsequent screening for chloramphenicol-resistant colonies on RCM agar plates containing 0.01 mg/mL chloramphenicol (Sigma) Further confirmation of mCherry-OVA expression was carried out by flow cytometry for mCherry expression (excitation at 545 ± 30 nm emission at 610 ± 75 nm) using the BD FACSAria III Fusion Cell sorter polymerase chain reaction and western blot Flow cytometry data was further analyzed using FCS Express version 5 software (De Novo) their origin of isolation and genome accession number Materials were directly adsorbed onto a carbon film membrane on a 300-mesh copper grid Grids were examined with Hitachi HT7700 electron microscope operated at 80 kV (Elexience – France) and images were acquired with a charge-coupled device camera (AMT) This work was carried out at the facilities and expertise of MIMA2 MET – GABI lactose or maltose (Sigma) without agitation A drop in OD values due to sedimentation is a characteristic of EPS non-producers Mice used for this study were 8–12 weeks old from a C57BL/6 background WT C57BL/6 mice for isolation of bone marrow and generation of BMDMs and BMDCs were purchased from Envigo (Oxfordshire OT-II mice were acquired from Jackson Laboratories (Bar Harbor United States) and breeding was maintained in house All mice were housed in UCC Biological Services Unit under specific pathogen free (SPF) conditions Standard housing conditions were maintained with 12 h darkness and 12 h light temperature controlled at 21°C and 50% humidity Animals were fed standard chow pellets and water was given ad libitum The animal work was performed in accordance with EU legislation in accordance with EU Directive 2010/63/EU for the protection of animals used for scientific purposes and approved by the Animal Experimentation Ethics Committee of University College Cork THP1-XBlueTM and THP1-XBlueTM-defMyD (MyD88 deficient) NF-κB reporter cell lines were purchased from Invivogen Cells were routinely cultured using Roswell Park Memorial Institute medium (RPMI 1640; Sigma cat R8758) supplemented with 10% Fetal Bovine Serum (FBS; Sigma) 100 μg/mL Normocin (Invivogen) and 200 μg/mL Zeocin (Invivogen) For the THP1-XBlueTM-defMyD cells 100 μg/mL Hygromycin B Gold (Invivogen) was added to the medium Cells were continuously maintained at a density between 7 × 105 cells/mL and 2 × 106 cells/mL NF-κB activity in THP-1 reporter cell lines was assayed using Quanti-blue (Invivogen cat Data was further analyzed using FCS Express version 5 software (De Novo) after an overnight culture of bacteria an OD600 nm of 1 corresponded to 109 CFU of bacteria Bacteria were washed twice in sterile PBS and resuspended in the appropriate mammalian cell culture medium with no antibiotics and concentration was adjusted as required For cytokine and NF-κB activity experiments BMDCs and THP-1 monocytes were seeded in 96 well plates at a density of 5 × 104 cells/well in RPMI with 10% FBS and incubated with bacteria at a multiplicity of infection of 10:1 bacteria:cells For BMDM and BMDCs experiments 100 ng/mL of LPS-B5 (Invivogen cat tlrl-b5lps) and 5 μM of ODN 1585 CpG (Invivogen cat tlrl-1585) were added as a positive controls Co-cultures were incubated for 24 h at 37°C in 5% CO2 cells were collected by centrifugation and supernatants analyzed for NF-κB activity (THP-1 cells only) For reverse transcription-quantitative polymerase chain reaction (RT-qPCR) BMDMs or BMDCs were seeded at 2.5 × 105 cells were seeded per well in 12 well plates in RPMI with 10% FBS and incubated with bacteria at a multiplicity of infection of 10:1 tlrl-b5lps) was used as a positive control in BMDM cultures and 5 μM of ODN 1585 CpG (Invivogen cat tlrl-1585) was used as a positive control in BMDCs cultures Co-cultures were incubated for 4 or 8 h at 37°C in 5% CO2 BMDMs were washed with ice cold PBS once and lysed using RLT lysis buffer (Qiagen) BMDCs were transferred to 1.5 mL tubes and centrifuged for 7 min at 400 × g at 4°C Supernatants were removed and cells lysed in RLT lysis buffer Both BMDM and BMDs lysates were immediately stored at −80°C until total RNA was extracted BMDM and BMDCs co-culture supernatants were analyzed for secreted cytokines TNF-α and IL-10 using mouse TNF-alpha DuoSet ELISA (R&D Systems cat DY410) and mouse IL-10 DuoSet ELISA (R&D Systems cat DY417) as per manufacturer’s instructions Supernatants from T cell and BMDM or BMDCs co-cultures were analyzed for secreted cytokines TNF-α and IL-2 using mouse TNF-alpha DuoSet ELISA (R&D Systems cat DY410) and mouse IL-2 DuoSet ELISA (R&D Systems cat DY402) as per manufacturer’s instructions TNF-α production by THP-1 reporter cell lines was measured in supernatants from these experiments using human TNF-α ELISA Duoset (R&D Systems cat Universal ProbeLibrary (UPL) probes and primers used for RT-qPCR bacterial cells were resuspended in ice-cold 10 mM Tris-HCl pH 8 transferred to tubes containing glass microbeads and bead-beated three times Lysates were centrifuged at maximum speed for 15 min at 4°C and supernatants were transferred to fresh 1.5 mL tubes Protein was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific) as per manufacturer’s instructions Fifty μg of protein for each sample was standardized and the appropriate volume of 4× LDS sample buffer and 10× sample reducing agent (Novex) was added to each sample Protein was denatured by heating at 70°C for 10 min Samples were separated on a 4–12% Bis-Tris Plus Gel (Invitrogen) alongside SeeBlue Plus2 Prestained Standard ladder (Invitrogen) and transferred onto a PVDF membrane (Millipore) for antibody detection Membranes were stained with Ponceau S to confirm protein transfer blocked for 1 h rocking at room temperature with 5% milk powder (Sigma) dissolved in 0.05% Tween-20 TBS (TBST) washed three times with TBST and incubated rocking overnight at 4°C with anti-OVA (Abbiotec cat Membranes were washed with TBST and incubated with HRP-conjugated goat anti-rabbit secondary antibody (Sigma) in 5% milk in TBST Membranes were washed with TBST and WesternBright substrate (Advansta) was added Images were acquired using Fujifilm LAS-3000 instrument and software 5 × 104 sorted BMDCs were seeded per well in 96 well plates in RPMI with 10% FBS but no antibiotics Bacteria expressing mCherry-OVA were added at ratio 10:1 OVA protein (Sigma) was added at a final concentration of 150 μg/mL as a positive control The cells were incubated with OVA protein or bacteria for 8 h at 37°C in a cell culture incubator at 5% CO2 CD4+ T cells were isolated from OT-II mice by spleen homogenization Red blood cells were lysed using RBC lysis buffer (Invitrogen) and CD4+ T cells separated magnetically using mouse CD4+ T Cell Isolation Kit (Miltenyi cat 130-104-454) as per manufacturer’s instructions Isolated T cells were then labeled with carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker kit (BioLegend cat 423801) as per manufacturer’s instructions After 8 h co-incubation with OVA protein or bacteria media was removed from the BMDCs cultures and fresh media containing 200 μg/mL gentamicin (Sigma) was added for 30 min at 37°C The cells were washed three times with PBS and fresh media was added along with CFSE-labeled CD4+ T cells from OT-II mice at a ratio of 1:1 T cells:BMDCs Plates were incubated at 37°C in 5% CO2 for 5 days T cells were then collected by centrifugation and stained with anti-mouse CD4 Brilliant Violet421 (BioLegend cat 135510) and anti-mouse CD25 R-phycoerythrin cyanine 7 (BioLegend cat T cell proliferation and activation was analyzed using the BD FACSCelesta Analyzer and data was further analyzed using FCS Express version 5 software (De Novo) 1 × 106 sorted BMDCs were seed in 6-well plates and co-cultured overnight at 37°C with B Cultured BMDCs alone were used as negative controls Cells were washed and incubated with gentamicin (200 μg/mL) for 30 min at 37°C and then washed 3 times in 1 × PBS and stained with CD45 APC fixed with 1% paraformaldehyde and re-suspended in 50 μl of wash buffer Fixed and stained cell samples were analyzed with an Amnis ImageStreamX MkII cytometer running INSPIRE software version 200.1.620.0 At least 2000 CD45 positive cells were acquired from each experimental sample Data was analyzed using IDEAS version 6.2.187.0 For every experiment at least three biological replicates with three technical replicates per experiment were performed Statistical analysis was compiled using one way ANOVA with Dunnett’s comparison in GraphPad Prism version 5.03 Statistical comparisons were completed using student’s t-tests and p-values less than 0.05 were considered significant homogenized before OD readings were taken from cultures over 6 h with no disturbance to cultures at all Bacteria were grown in media containing either glucose Data show the average of three independent experiments there was no correlation between EPS status and immune activation of human THP-1 monocytic cells breve strains are MyD88-dependent but EPS-independent breve strains were incubated with THP1-XBlue and THP1-XBlue-defMyD reporter cell lines for 24 h (A) NF-κB responses were assessed by measuring reporter gene production (B) TNF-α secretion was assessed by ELISA Data shown is the average of three independent experiments performed in triplicate wells Statistical analysis was assessed using the student t-test in GraphPad Prism where *p-value < 0.05 and ***p-value < 0.0005 Transmission electron microscopy images of B Opposing effects of EPS on murine macrophage cytokine responses to B (A,B) Gating strategy to identify and sort BMDMs The bone marrow cells were analyzed and sorted by multi-color flow cytometry CD11b contour plots were obtained by gating on MHC-II+CD11c+ cells CD115 contour plots were obtained by gating on MHC-IIlowCD11bhigh cells CD115+ cells were sorted and considered as pure macrophages population CD115 contour plots were obtained by gating on MHC-IIhighCD11blow cells CD115– cells were sorted and considered as pure dendritic cells population (C,D) TNF-α and IL10 secretion from BMDMs co-cultured with B JCM7017 and their isogenic EPS– mutants (E,F) TNF-α and IL10 secretion from BMDCs co-cultured with B Data is the average of three independent experiments performed in triplicates Statistical analysis was assessed using the student t-test in GraphPad Prism where *p-value < 0.05 and **p-value < 0.005 this gene expression data indicate that the presence of EPS suppresses the expression of cytokine and co-stimulatory genes required for activation and maturation of dendritic cells in response to B EPS modulates the expression of immunomodulatory genes in BMDCs Sorted BMDCs were co-cultured or not [untreated (Unt)] with B breve strains as indicated (CpG was used as a positive control) for 4 h RT-qPCR was carried out and the expression of (A–F) cytokine genes (G–I) antigen presentation and co-stimulatory genes and (J–M) tolerogenic genes were assessed Expression changes are relative to β-actin Gene expression differences between the groups were compared first with the untreated control Different letters (a and b) indicate statistical difference for each presented gene expression Letter a indicates no statistical difference between the indicated group and the untreated control letter b indicates significant differences between the indicated group and the untreated control (p < 0.05 of Dunnett’s test) the student t-test was used to compare wild type Bifidobacteria (both UCC2003 and JCM7017) with their isogenic EPS mutants (*p-value < 0.05 and **p-value < 0.005) these results indicate that while EPS does not affect the uptake of bacteria by DCs it does reduce processing and presentation of bacterial derived OVA antigen and activation of antigen specific CD4+ T cells by DCs EPS protects from antigen presentation of B (A) mCherry expression (pMG-mCherry OVA) in the indicated B Histograms were obtained by gating on bacterial cells Black and gray histograms were obtained from parental B breve UCC2003 and JCM7017 wild type or EPS– Red and blue histograms were obtained from B breve UCC2003 EPS– transformed with the plasmid pMG-mCherry OVA Purple and green histograms were obtained from B breve JCM7017 EPS– transformed with the plasmid pMG-mCherry OVA (B) Western blot for OVA protein production by pMG-mCherry-OVA and pMG-mCherry transformed strains of B 0.025 μg of ovalbumin was used as a positive control (C,D) Growth curves of strains transformed or untransformed with the pMG-mCherry-OVA plasmid over 24 h Growth curves are representative of three experiments (E) Schematic of the antigen presentation assay setup (F) Sorted BMDCs were co-cultured or not with different bacterial strains expressing or not OVA (as indicated) for 8 h mCherry only expressing bacteria were used as negative controls Cells were then co-cultured with OT-II CD4+ T cells for 5 days and analyzed by multi-color flow cytometry Percentage of proliferated CD4+CD25+ OT-II T cells was quantitated and presented as mean ± SEM Data is the average of three independent experiments performed in triplicate wells we report on the production and immunomodulatory effects of EPS from a collection of human-isolated B EPS was produced differentially across a panel of strains and EPS status did not correlate with obvious immunomodulatory effects in a series of immune cell-based assay systems breve EPS was required for suppression of gene expression associated with and required for full maturation of DCs EPS was necessary to prevent processing and presentation of antigen from the strains and antigen-specific activation of CD4+ T cells by DCs we have identified a new immunomodulatory role for EPS which indicates that EPS is utilized by B breve for immune evasion perhaps as a mechanism to promote host-microbe mutualism This variability could also be altered by growth in different media but the mechanistic reasons for this have yet to be explored and represent a pure population of DCs with no contaminating macrophages This might explain why they did not show an obvious inflammatory response to our B breve JCM7017 did not induce expression of the Il12b/IL12p40 subunit in BMDMs implies that the bacteria may not support differentiation of TH1 or TH17 T cells It also implies that EPS is responsible for controlling this as the EPS– mutant upregulated the expression of Il12b/IL12p40 in BMDMs Our study shows that while EPS production was heterogeneous amongst human-isolated B breve UCC2003 and JCM7017 had similar effects on maturation and antigen presentation function of BMDCs It is important to acknowledge that the biochemical composition and density of EPS observed in both WT strains may contribute to the differences observed in BMDM and BMDCs immune responses molecular and cell-based investigations are required to understand the biochemistry and immunological effects of different EPS structures and their effects on dendritic cell biology Our observations suggest that EPS may contribute to an important immunomodulatory effect of B breve strains by blocking activation and antigen presentation function of DCs This would allow these strains to manipulate DCs for immunomodulatory effects promoting immune evasion and host-microbe mutualism by these gut symbionts The original contributions generated for this study are included in the article/Supplementary Material further inquiries can be directed to the corresponding authors Use of animals for this study was reviewed and approved by Animal Experimentation Ethics Committee of University College Cork and FB: methodology and writing – original draft and KN: writing – review and editing All authors contributed to the article and approved the submitted version This work was supported by grants from Science Foundation Ireland – namely a research center grant (SFI-12/RC/2273) to APC Microbiome Ireland OH is employed by the company Luminex Corporation which manufactures the Amnis ImageStream flow cytometer used in this study We would like to acknowledge and thank the Flow Cytometry Platform APC Microbiome Ireland located at University College Cork for assistance with flow cytometry analysis The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.653587/full#supplementary-material Extraction of the same novel homoglycan mixture from two different strains of Bifidobacterium animalis and three strains of Bifidobacterium breve Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp Comparative genomics and genotype-phenotype associations in Bifidobacterium breve Google Scholar Comparative genomics of the Bifidobacterium breve taxon Exopolysaccharides synthesized by Bifidobacterium animalis subsp lactis interact with TLR4 in intestinal epithelial cells “Biological activities and applications of Bifidobacterial 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function via STAT3 signaling pathway Rossini V and Nally K (2021) Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells Copyright © 2021 Hickey, Stamou, Udayan, Ramón-Vázquez, Esteban-Torres, Bottacini, Woznicki, Hughes, Melgar, Ventura, Van Sinderen, Rossini and Nally. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Valerio Rossini, dmFsZXJpby5yb3NzaW5pQHVjYy5pZQ==; Ken Nally, ay5uYWxseUB1Y2MuaWU= †These authors share senior authorship Get our news on your inbox! Suscribe x MercoPress, en Español Montevideo, May 5th 2025 - 11:26 UTC Many young people in Italy are expressing outrage on social media after a judge cleared a school caretaker of groping a teenager because it did not last long enough The case involves a 17-year-old student at a Rome high school She described walking up a staircase to class with a friend a hand touching her buttocks and grabbing her underwear you know I was joking,” the man told her when she turned around He admitted to groping the student without consent A Rome public prosecutor asked for a three-and-a-half year prison sentence but this week the caretaker was acquitted of sexual assault charges what happened “does not constitute a crime” because it lasted less than 10 seconds palpata breve - a brief groping - has become a trend on Instagram and TikTok in Italy The videos are often uncomfortable to watch but they have the aim of showing just how long 10 seconds can feel The first was posted by White Lotus actor Paolo Camilli and since then thousands of people have followed suit Another video was reposted by Chiara Ferragni Italy's most famous influencer who has 29.4 million followers on Instagram He goes on to say that the judges' decision to acquit the caretaker shows just how normalized sexual harassment is in Italian society A post on the Freeda Instagram account says: “This sentence is absurd The duration of the harassment should not diminish its severity.” performing an “awkward manoeuvre without lust” it was no joke to me,” the student told Corriere della Sera newspaper “The caretaker came up from behind without saying anything He put his hands down my trousers and inside my underwear he pulled me up - hurting my private parts This is not how an old man should 'joke' with a teenager.“ ”That handful of seconds was more than enough for the caretaker to make me feel his hands on me.“ She says she feels doubly betrayed - by her school and by the justice system ”I'm starting to think I was wrong to trust the institutions The student fears the judges' ruling will deter girls and women from coming forward if they are subjected to such attacks Recent figures from the EU's Fundamental Rights Agency (FRA) suggested that 70% of Italian woman who had suffered harassment between 2016 and 2021 did not report the incident “They will feel that reporting abuse is just not worth it because silence protects the aggressors.” Commenting for this story is now closed.If you have a Facebook account, become a fan and comment on our Facebook Page