Volume 7 - 2020 | https://doi.org/10.3389/fcvm.2020.00119 Parts of this article's content have been modified or rectified in: Erratum: Advances in IVUS/OCT and Future Clinical Perspective of Novel Hybrid Catheter System in Coronary Imaging Intravascular ultrasound (IVUS) and optical coherence tomography (OCT) have been developed and improved as both diagnostic and guidance tools for interventional procedures over the past three decades IVUS has a resolution of 100 μm with a high tissue penetration and capability of assessing the entire structure of a coronary artery including the external elastic membrane whereas OCT has a higher resolution of 10–20 μm to assess endoluminal structures with a limited tissue penetration compared to IVUS integrated IVUS and OCT into a single catheter system With their inherent strength and limitations the combined IVUS and OCT probes are complementary and work synergistically to enable a comprehensive depiction of coronary artery we summarize the performance of the two intracoronary imaging modalities—IVUS and OCT—and discuss the expected potential of the novel hybrid IVUS–OCT catheter system in the clinical field The two modalities of imaging have advantages and disadvantages which are described in the next chapter (1-2) Timeline of intravascular ultrasound and optical coherence tomography with advances of PCI era A history focused on intracoronary imaging devices (IVUS and OCT) Red and blue frames indicate representative events associated with IVUS and OCT Society for Cardiovascular Angiography; ESC European Association for Cardio-Thoracic Surgery; MACE Those results support the use of IVUS as a PCI guidance tool in the contemporary PCI era IVUS and OCT have evolved in parallel over the last years. Recently, however, hybrid IVUS-OCT systems were developed to merge the advantages of both modalities into a single catheter (28–30) This review aims to summarize the differences and complementary aspects of IVUS and OCT and describe the novel hybrid IVUS–OCT catheter systems Since this paradox was possibly due to longer procedural time for stent optimization in the IVUS-guided PCI group operators should pay attention to the use of contrast during IVUS-guided PCI as well Observational abilities of IVUS and OCT in the settings of several intracoronary structures IVUS indicates both grayscale IVUS and IVUS with radiofrequency analysis The calcium score is composed of maximum angle of >180° (2 points) maximum thickness of >0.5 mm (1 point) The calcium score of 4 was significantly associated with stent underexpansion when compared to a score of 0–3 (78 vs suggesting heavily calcified lesions that need debulking it will be important to assess remodeling patterns at long-term follow-up after the full absorption of the deployed scaffolds it also takes time and effort to combine the two imaging modalities into a single image a fact that makes impossible the broad application of this approach in research A hybrid IVUS–OCT catheter system is warranted to enable accurate online coregistration and fusion of high-quality images obtained by the two imaging probes during a single pull-back Various types of arrangements of IVUS and OCT transducer There were three types of arrangements of IVUS and OCT transducer; (A): back-to-back arrangement; (B): coplanar arrangement; (C): sequential arrangement; (D): colinear arrangement The colinear arrangement can acquire the strictest coregistration image of IVUS and OCT in three types of arrangements the first clinical use of the hybrid IVUS–OCT catheter was reported by Sheth et al. showing beautiful coregistered images with clinically acceptable specifications with respect to the size Pullback speed can be selected from 0 (manual control) and 25 mm/s with a maximum pullback length of 100 mm the pullback will be performed with a frame speed of 100 fps after blood clearance The system does not have a setting that allows for the acquisition of OCT only anticipated disadvantage to collecting IVUS at the same time The maximum field of view radius is 6 mm derived from IVUS CONAVI Novasight hybrid imaging catheter; external appearances The specifications of CONAVI Novasight Hybrid and TERUMO Dual Sensor system CONAVI Novasight hybrid imaging catheter; sample images (A) Coregistered intracoronary images of superficial atheroma in IVUS (A-1) and OCT (A-2) (C) Measurements of lumen size by the coregistered image of IVUS and OCT distances) made in an IVUS image are automatically copied over into the OCT image and vice versa The first clinical usage was reported by Sheth et al. in 2018 (88) the Novasight system could provide coregistered and co-aligned IVUS and OCT images in a patient with recent ST-segment elevation MI (STEMI) followed by PCI for a non-culprit lesion of the LAD and deeply embedded tissues were more clearly identified by IVUS images than OCT and fine dissections were more clearly identified by OCT imaging The Novasight system is currently FDA 510(k) cleared and has Health Canada approval A prospective observational study using the Novasight hybrid imaging catheter has been completed and demonstrated its feasibility and efficacy for diagnostic purposes and PCI guidance in 20 patients with a chronic coronary syndrome or ACS (NCT03484975) Online OFDI 3D reconstruction will facilitate comprehensive evaluation of complex coronary artery structures TERUMO hybrid IVUS–OCT catheter system; external appearances TERUMO hybrid IVUS–OCT catheter system; sample images (A) Coregistered intracoronary imaging of thrombus in IVUS (A-1) and OCT (A-2) in a cadaver coronary artery (B) Coregistered intracoronary imaging of calcification in IVUS (B-1) and OCT (B-2) in a cadaver coronary artery The cost is expected to be similar to that of OFDI The quantitative analysis of plaque burden would be well-assessed by IVUS, especially by IVUS-RF analysis. Recently, near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) was introduced to more accurately detect lipid core plaques (112–114) Futures studies are expected to examine the efficacy of the combined IVUS–OCT imaging catheters in detecting vulnerable plaques The hybrid IVUS–OCT catheter also could allow one to make a more accurate coronary artery model and may provide new insight into the association between wall shear stress and a plaque progression/regression Hybrid IVUS–OCT catheters have a high potential to support procedures in a wide range of circumstances by its high diagnostic efficacy derived from the combination of the two imaging modalities the hybrid catheter is expected to be widely utilized in daily clinical practice we describe the potential value of this hybrid catheter in guiding PCI in the most challenging lesions such as LMCA disease and multivessel coronary artery disease (MVCAD) the rate of ischemia-driven revascularization was significantly higher in the PCI arm than in the CABG arm at 5 years (16.9% in the PCI arm and 10.0% in the CABG arm Although IVUS guidance was strongly recommended in this trial it was used only in 77.2% of the cases in the PCI arm we can use each function according to the process comprehensively while IVUS will be utilized for the indication and/or deciding the location of the stent landing zone PCI could become equivalent to CABG only in cases fulfilling these criteria in the field of MVCAD and the use of an intracoronary imaging is mandatory in this setting MVCAD often shows a mixture of various types of lesions (e.g. different strategies might be required according to each lesion characteristic The hybrid IVUS–OCT will be able to assist operators to tailor treatment strategy according to lesion types intracoronary imaging-derived FFR may be used to assess lesion severity and identify those that need treatment and estimate the residual ischemic risk after stent implantation which is also one component of “best practice PCI” for MVCAD The hybrid catheter has a marginal incremental cost to build relative to a single modality imaging catheter that would not greatly impact the overall cost-effectiveness of intravascular imaging The consoles and patient interface modules have costs that are associated with supporting ultrasound electronics as well as the optical components for OCT incremental cost over single modality systems to provide this advanced dual modality imaging capability For the purpose of detecting a high-risk plaque, a recent LRP trial demonstrated the impact of a Lipid Core Burden Index derived from NIRS-IVUS on future cardiac events, which was independent of intravascular ultrasound plaque burden or minimum lumen area (113) Further clinical trials will be needed to compare the hybrid IVUS-OCT system vs NIRS-IVUS in terms of the prognostic performance in patients at risk The hybrid IVUS–OCT system could be the answer in the near future and contributed to all revisions of the manuscript and MT gathered and reviewed the literature and contributed to all revisions of the article and CB gathered and interpreted the literature and contributed to all revisions of the manuscript as experts of the field IM and BC gathered and interpreted the data and contributed to revisions of the manuscript All authors provided final approval of the version to be published IM was employed by the company Terumo Corporation BC has the following conflicts of interest with Conavi Medical Inc.: co-founder JP reports personal fees and non-financial support from Philips/Volcano 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 computed tomography coronary angiography; DES European Association of Percutaneous Cardiovascular Interventions; EEM major adverse cardiac and cerebrovascular events; MI Balloon angioplasty - the legacy of andreas gruntzig MD 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January 2022 this collection feels as timeless as the eateries whose stories it tells Ask Faye Hara and her family what they do at Sekiya’s Restaurant and they’ll chuckle “What don’t we do?” she and her son-in-law Trey Paresa say Part of the third and fourth generations to run the restaurant across from Kaimukī High School they do everything from payroll and filling orders to washing dishes Deanna Hara—the great-granddaughter of founders Taisuke and Katsuko Sekiya—can step away from her marketing and social media duties to fry up tempura “She doesn’t like it,” mother Faye says with a laugh Since the Sekiyas took over an okazuya in a single-story building on School Street then eventually bought the building on Kaimukī Avenue in 1955 from creating the house-made dashi for nabeyaki udon and saimin to maintaining the dining room and the koi pond outside The classic Japanese menu retains most of the originals—Faye says a longtime customer recently brought in a menu he found from Hawai‘i’s territorial days “and it’s pretty much the same”—down to the oxtail soup recipe that came from a Chinese neighbor of one of the Sekiyas’ daughters oyako donburi and a list of drinks from the soda fountain: vanilla and chocolate Cokes One of the biggest challenges is staying consistent as longtime businesses and vendors close Meadow Gold stopped making the sherbet Sekiya’s used for Orange Freeze forcing Sekiya’s to shrink its once extra-large cone sushi to fit the new they make as much as possible in-house the way it’s been done since the 1940s: marinating chicken thighs before enveloping them in an impossibly thin crispy coating and rolling potato hash into balls and dunking them in fluffy tempura batter The loyal group of longtime Sekiya’s customers are quick to tell the folks there whenever something tastes different “We have regulars who come in every day,” Paresa says “Some of them only come in for a saimin and a mac-potato salad It’s not only our family working for generations our customers come in for generations.” —CY 2746 Kaimukī Ave., (808) 732-1656, sekiyasrestaurant.com, @sekiyasrestaurant people lined up at Forty Niner for what they thought would be a last saimin and cheeseburger deluxe a chance to sit on the stools and eat at tables that had been there since brothers Richard and Henry Chagami returned from their tour with the 442nd Regimental Combat Team and opened a restaurant Richard retired that year and passed the historic building “I knew there would be a lot of eyes on me,” Cordes says “People have been bringing their families They were going to know the menu and taste of the food better than I did.” after a few months of much needed renovations—the kitchen had a single burner and no telephone—he reopened with Richard’s sister saimin dashi and all the original menu items Richard would even stop by now and then: “He would just say expanded the menu to breakfast with pancakes waffles and French toast and rotating specials throughout the day Longtime diners can now dig into the Forty Niner pancakes—which Cordes says were the first to feature haupia sauce and macadamia nuts—banana French toast chilaquiles with eggs and garlic chicken while sitting on the eatery’s original stools “Our restaurant was built with a saimin and a burger and that’s withstood the test of time,” Cordes says 98-110 Honomanu St., ‘Aiea, (808) 484-1940, fortynineraiea Tracking down the fabled story about the Halekūlani’s almost century-old restaurant and the book that first gained it worldwide fame is like reading a detective novel The tie between author Earl Derr Biggers and the Waikīkī home that bore the same name is more nebulous Some claim that’s where he heard stories of Other accounts say he stayed somewhere else and only read about Apana’s cases in newspapers in Boston when Clifford Kimball acquired Brown’s place with the wraparound lānai for the Halekūlani Hotel in 1926 and the new building included a rebuilt House Without a Key that offered sunset cocktails on the terrace and upscale continental fare diners could order a teriyaki tenderloin steak with white rice and multiple sides for $3 then finish with a whole baked pineapple “in flames.” The dishes and dining room have changed over time most recently when the hotel closed for renovations during the pandemic But when you step into the shaded bar or refurbished restaurant you’ll still be able to get a signature mai tai and a slice of coconut cake 2199 Kālia Road, (808) 923-2311, halekulani.com, @halekulanihotel and a jumble of painted and letter-board menus track the dishes people have returned to for generations: saimin I found it this way eight years ago after my sister mentioned that she liked the tofu salad but the only time Jane’s Fountain crept onto my radar was when my piano teacher entered her students in a contest for tickets to the Honolulu Symphony My mother dragged me to Blaisdell Concert Hall and tried to cheer me with the promise of a treat at Jane’s Fountain Jane’s Fountain is a vestige of Liliha’s heyday when trolleys ran up the hill from King Street and businesses followed The first L&L Drive-Inn opened down the block the original Liliha Bakery was practically next door and Young’s Fish Market shared the same building as Jane’s And it’s been decades since there was a soda fountain there: You can see its ghost in the round imprints of counter stools running in a neat line along the floor near an ancient Coca-Cola dispenser (“Refresh Yourself,” it says “Have a Coke”) that holds pale gold pomelos for a Chinese altar above Jane’s Fountain now is the domain of Karen Kan who bought it from the Nakasone family in 2010 For 10 years after she arrived from southern China ran next door to tend to her Jane’s Boutique dress shop leaving Kan in charge to speak with customers Kan’s menu additions were simple things like chicken katsu and shrimp canton with sweet-sour sauce including cheese sandwiches and corned beef with onion Kan knows my order now: pork squash stir-fry and steaming shrimp-filled sari sari or chopped steak with tomatoes Though a food snob the first time I walked in I’ve come to love Jane’s Fountain for what it is—a no-frills place of feel-good memories and flavors whether it’s saimin and a cheeseburger or what it was for my mom which I finally understand was the comfort she was offering me the story of Natsunoya Tea House is like the clouds that flit across ‘Ālewa Heights—rich white tufts that fade into wisps the original wooden buildings have hosted wedding banquets graduation parties and kanreki 60th birthday celebrations There’s even a sushi bar tucked in the depths Natsunoya drew visits from John Wayne and other A-list celebrities Other teahouses—Kanraku by Kapālama Canal Rainbow Garden in Mō‘ili‘ili Mochizuki Tea House on Kunawai Lane—once dotted hillsides where streams and springs flowed through Japanese neighborhoods but now Natsunoya is the only local-style teahouse left “It was a place where Japanese could come and cut loose in a sense,” says third-generation owner Laurence Fujiwara Jr “Some guests would take baths in the furo with Hawai‘i’s Japanese-speaking population growing Fujiwara’s grandfather carved out a road up ‘Ālewa Heights installed telephone poles and built a teahouse called Shunchoro Shuichi Fujiwara would add tatami mat-lined cottages for private dinners The bath was where the sushi counter is now (it’s still called the furoba Natsunoya’s unlikely location can be attributed to its stunning view—it takes in nearly all of O‘ahu’s south shore The Fujiwaras had a telescope through which they invited guests to admire the view a Japanese spy posing as a consulate officer habitually trained it at the sprawling naval base the Red Cross requisitioned the teahouse buildings with the future H-1 freeway set to run behind Foster Botanical Garden and right through the teahouse owned by Laurence Fujiwara Sr. the younger Fujiwara moved his operation up the hill to Shunchoro and renamed it Natsunoya diners with more spartan palates ate grilled fish and nishime; today spicy ‘ahi rolls and local-style tempura cocooned in thick survives on the banquet menu in salad form stuffed with haupia and peanut butter and dusted in coconut flakes “You have to change with time,” Fujiwara says you get eaten up and swallowed up and spit out But we’re going to keep the tradition of it.”—MT 1935 Makanani Drive, (808) 595-4488, natsunoyahawaii.com, @natsunoyateahouse whether it was chef Mark Noguchi or chef Sheldon Simeon may well have been talking about Palace Saimin The first was in 2009 when Setsuko Arakaki a longtime waitress there who bought the place from founder Kame Ige in 1975 and concerned employees asked Arakaki’s daughter looked at what if we didn’t,” says Scott Nakagawa Scott was a business consultant with a degree in architecture They took over a saimin stand where not much had changed since before they were born Seven tables sat in a two-story cinderblock walkup Palace’s second location; the original site was at Beretania and Ke‘eaumoku streets A Coca-Cola letter-board menu listed saimin the noodles nestled in Ige’s original broth of pork bones and dried shrimp Thick skewers of char-grilled beef dripped with old-school teriyaki sauce made a beeline to the water fountain to fill their own cups yelled their orders into the kitchen and sat down It went against Scott’s instincts to not transform the place into a cool midcentury saimin house So the Nakagawas painted the walls and standardized recipes and hours (Arakaki would open for lunch personalized attention she used to give first-graders on customers and employees Business plummeted after O‘ahu’s first lockdown; the second brought Palace to its knees Hardly anyone thought of saimin for takeout the Nakagawas calculated how much longer they could stay open and still help furloughed employees and mapped out the end Then a local TV news team heard about their plight The segment drew lines and a stream of takeout orders Regulars came back and brought new customers Nothing in Scott’s years of consulting prepared him for the epiphany that simple bowls of saimin really are life—a vital and vulnerable part of local life customers collected and dropped off cardboard boxes (they still do) a customer retrieved parts from his car and fixed it It touches the heart of a small business.” —MT A sign has lit up the intersection of Gulick and Kalihi streets for almost half a century The simple message tells diners exactly what they’ll find inside: Tasty Chop Suey Hung Pui Wong sailed from China to Hawai‘i in 1939 and worked in restaurants then as a cook with the 298th Infantry Regiment in World War II He and his wife opened their own place on King Street and moved to the current spot in 1963 when people ordering wonton soup could watch workers wrap the dumplings at the table next to them you can still find piles of snow peas being cleaned on a tabletop next to the Pepsi refrigerator that replaced the glass bottle Coke vending machine that served customers through the ’80s And owner Shirley Ho still sells steaming hot melamine bowls of wonton mein along with $3 bags of shrimp chips and rice cake in knotted plastic bags near the cash register it seems fitting that its fortune has often turned with the tide Co-founder Annette La Mariana Nahinu arrived in the Islands with her new husband on a sailboat in the 1950s they leased 5,000 square feet of swampland near Ke‘ehi Lagoon and built a marina themselves A young George Ariyoshi recalled seeing her at that time “a young haole woman with a wheelbarrow pushing dirt and working so hard,” he told the Honolulu Star-Bulletin in 2008 was damaged in a tsunami three years later then was forced out when the state canceled Nahinu’s lease in 1975 every blade of grass and tree she had planted—including an 18-foot-tall shower tree—just down the shoreline the place she built survived a legal battle over ownership another tsunami and a new threat to its state lease La Mariana’s restaurant remained blissfully in the ’50s Diners sit in rattan chairs under fishing nets and floats illuminated by twinkling Christmas lights ordering French onion soup and poke while sipping on cocktails with tiny parasols and spiked with rum When the musicians aren’t crooning lounge tunes with longtime employee Judith Calma at the helm and a lease extension that stretches to 2040—as well as some Hollywood attention as the namesake of a tiki bar on Magnum P.I.—it should have been smoother sailing the restaurant decided to close for two weeks which then stretched into more than 18 months 50 Sand Island Access Road, (808) 841-2173, @lamarianasailingclub the ashes of Susumu and Ellen Nakagawa are in a glass cabinet near the entrance of the restaurant “They’d been at the restaurant for so many years I thought I’d put them in the place they love the most,” their son Colin says their spirit is still helping in the restaurant.” serving the same chicken and mullet recipes from when the original Seaside Club opened in 1915 The Seaside Club was initially a private resort with a swimming pool for members and fishpond for the mullet took it over in 1926 and opened it to the public The 1946 Hilo tsunami destroyed the original building; Seiichi and his wife rebuilt across the street next to a larger 30-acre fishpond the same: American-style food including steak; Seaside Club’s chicken; and fish from the ponds and mullet steamed in ti leaves with just rock salt But Susumu tinkered elsewhere: His friends from the Army’s 442nd Regimental Combat Team helped him build rock walls in the fishpond as holding pens for the fish he raised—at one point the retired USDA entomologist cultivated seven different varieties including rainbow trout When Colin returned to Hilo from Seattle in 1983 to join the family business he introduced Pacific Rim dishes and added dining rooms overlooking the water the aqua farm at Seaside Restaurant and Aqua Farm is less of a focus falling to the wayside when Susumu could no longer physically manage it But Colin continued to bring him to the ponds to rest in the gazebos on the artificial islands one of the casualties of the outbreak at the Yukio Okutsu State Veterans Home.) Colin also keeps his dad’s legacy alive by keeping some fish in the pond prepared just like it was at the Seaside Club more than a century ago 1790 Kalaniana‘ole St., Hilo, Hawai‘i Island, (808) 935-8825, seasidehilo.square.site Café 100 survived two tsunamis: the first in 1946 three months after Richard and Evelyn Miyashiro opened the original diner three weeks after they moved into a newly built restaurant down the street From his home behind the new Café 100 Richard watched the waves pulverize his dream restaurant but bearing the brunt of the water’s force it likely saved his and his family’s lives Richard went from a dine-in restaurant to the drive-in concept still in operation today named in tribute to the 100th Infantry Battalion that Richard was part of Richard was one of the first to put an egg on a hamburger patty the restaurant is even trademarked as “The Home of the Loco Moco.” Café 100 served 10,000 loco mocos a month—there are now 30 varieties mounded with chili (which Richard’s daughter who works days at his family’s longtime Chinese restaurant There may be many more loco mocos on the menu now Leung retains her grandparents’ philosophy of “fresh food at reasonable prices.” — MC 969 Kīlauea Ave., Hilo, Hawai‘i Island, (808) 935-8683, cafe100.com, @cafe100hilo People wondered if Teshima Restaurant could exist without its namesake she took the restaurant back—“It was just declining,” remembers her great-granddaughter Noelani Greene—and worked for another four decades Even after she finally stepped away from the kitchen at the age of 105 She spent her last year in her home a few steps behind the restaurant before passing away in 2013 People still come for favorites like the No sukiyaki and the restaurant’s signature shrimp tempura in a lacy A woman waiting in the pickup line says she has been coming here for 40 years and remembers “Grandma Teshima” always bringing her green tea ice cream and stopping to chat Stories invariably include Teshima’s graciousness and generosity of attention She sometimes entertained celebrities—James Stewart was a regular when he had a ranch in South Kona in the ’60s—but you didn’t have to be one for her to come to your table with a piece of maki sushi and a smile she’s always scolding us,” Greene says You’re not presentable.’” Teshima grew up in her parents’ store in Honalo But “it was kinda boring,” she said in a Kona Historical Society interview she began bringing in 100 pounds of ice from Hilo daily to make ice cream at night to sell the next day (The ice house is still on the property.) Next she installed a soda fountain and a U-shaped bar; then she tore down the store and built a 200-plus-seat restaurant bringing in a Japan-trained chef to create the menu that’s mostly in place now who started as a dishwasher about 40 years ago who manages the restaurant and works part time as a dental hygienist took advantage of the temporary closure to renovate the interior She reupholstered the booths and replaced the floor but otherwise tried not to change too much “Legacy is a big thing,” Greene says “I feel like if I don’t fulfill it or people say you’re changing everything,’ that really would break my heart.”—MC 79-7251 Hawai‘i Belt Road, Kealakekua, Hawai‘i Island, (808) 322-9140, teshimarestaurant.com It built generations of fans on a stack of fluffy dinner-plate-size pancakes Wailuku landmark Tasty Crust has changed hands a few times: Joe Kozuki opened Tasty Crust bakery in the 1940s just around the corner from Wailuku Sugar Mill whose sister created that secret hotcake recipe then Mike and Patsy Takaoka took over in 1957 and ran it for decades a second and third generation of Takaokas are in charge his oldest daughter Tammy Roloos and youngest son Brandon Carvalho still serve saimin with won bok red sweet-and-sour spareribs and those world famous hotcakes,” as it says on the 50-year-old sign and as soon as the waitstaff see the guy pulling into the parking lot he just goes right to his seat,” Curtis says That’s the great part.” —CY Image: Courtesy of Honolulu Star-Advertiser 1770 Mill St., Wailuku, Maui, (808) 244-0845, @tastycrustmaui a manju recipe and a Chinese cook created the institution that keeps people lining up in Wailuku Sam Sato left his plantation job at 19 to open a store in Spreckelsville Middle Village Three with mom Mite Sato and sisters next opened in the sugar town of Pu‘unēnē in 1966; the cook there helped create Sato’s signature soupless dry noodle recipe The sugar camps started closing in the ’70s this time back to Wailuku and Sato’s current space in the Wailuku Millyard Sato’s grandson Kirk Toma keeps things going as people line up for bowls of chewy noodles topped with char siu bean sprouts and green onions with dashi on the side; teri burgers; and Mite’s lima bean or red bean manju and the pork chops had bewitched the woman all night she told the waitress at Manago Hotel that she had had them for dinner the previous night Gourmet magazine highlighted the Manago Hotel’s golden-fried pork chops in an article recognizing 20 legendary restaurants across America The restaurant has now outlasted the magazine it’s one cook’s sole job to prepare the pork chops which are fried in a dedicated cast iron pan made by Hilo Iron Works in the 1920s borrowed $100 to buy a small house in South Kona salesmen and taxi drivers traveling between Hilo and Kona were asking to spend the night The small home-turned-hotel expanded in 1929 with a second floor of rooms and the restaurant started serving full meals and sake the Army contracted Manago Hotel to feed soldiers staying at Konawaena High School a place where reservations are still recorded by hand on a set of clipboards At the restaurant entrance is a porcelain hand sink that coffee farmers once used to wash the dirt off their hands No one seems to know when the udon was retired and the pork chops added the menu is largely unchanged since the 1940s: a dozen items including liver and onions hamburger steak and small local fish such as ‘ōpelu or akule pan-fried simply in butter and accompanied by lemon wedges and tartar sauce it came with a large bowl heaped with rice and side dishes on little melamine plates steamed vegetables and a limu namasu that I now think should be a de facto condiment alongside shoyu Britney Manago grew up living below the kitchen and “eating the same thing over and over again—mahimahi and the pork chops.” She went to college in California then homesickness drove her back to the family business “This is the only thing I’ve ever really known that I’ve loved and wanted to always be a part of.” —MC 82-6155 Hawai‘i Belt Road, Captain Cook, Hawai‘i Island, (808) 323-2642, managohotel.com he wanted to make just a few small changes He attempted to switch the lighting fixtures One of his longtime customers advised against it He thought about trying a different coffee who drove 40 miles from Waimea to Līhu‘e every day for breakfast opened more than a century ago has survived two hurricanes the Great Depression and the Great Recession Ota laughs affectionately and says: “You gotta be careful when you change things.” Since Denjiro left his job as a cook to open the first coffee shop in Līhu‘e a chef who took over in 1925 at the original Tip Top Building and launched many of the restaurant’s signature recipes Mitchell later moved the shop to its current location and expanded it to include a motel and bar helped out in between assignments with the Air National Guard 1938 Tip Top Café ad in the Honolulu Advertiser Not all of the younger Ota’s updates were rejected by regulars The accounting graduate from UH Mānoa streamlined and set a new direction for the business side even after Hurricane ‘Iniki blew the rooftop off the hotel in 1992 the construction crews and the insurance company agreed to restore the café And as Ota and his neighbors cleaned up debris Tip Top’s chefs fired up the gas burners and cooked free food for the Air National Guard members who arrived to help and anyone else who needed a hot meal Ota decided to rent the bakery to outside vendors And the macadamia nut cookie that the restaurant proudly claims as the first in Hawai‘i—Ota’s grandfather “was experimenting with walnuts but they were too oily so he switched to macadamia nuts,” Ota says—is no longer on the menu But other items from the days when plantations and vast cane fields dominated the landscape remain and are cherished are from the original top-secret recipe and process of Ota’s grandfather The only change is that it’s now available all day Some grow up and even have jobs here.” 3173 Akahi St., Līhu‘e, Kaua‘i, (808) 245-2333, tiptop-motel.com ‘Aiku‘e and Kekahu Napoleon​-​Ahn opened a coconut-based ice cream shop They didn’t expect to spend more of their time making kūlolo The second annual celebration of Hawai‘i’s Latino community showcases all kinds of food Here’s what you can expect at the new budget KBBQ spot With a message of solidarity after last weekend’s tragedy in Vancouver the Fiesta will be a show of cultural pride 80-plus ways to pamper Mom’s taste buds on Sunday 25 years after he made his professional debut, Ono led Consadole Sapporo out one last time in their J1 League season finale -- poetically against Urawa Red Diamonds who he debuted with -- before coming off in the 22nd minute to a rapturous ovation at Sapporo Dome His swansong was to be his sole league appearance in 2023 He also featured in the one league game last term Having played just 35 league minutes in these past three seasons it has been apparent for some time now that Ono's storied career was slowly but surely coming to an end and yet that hardly means he has not contributed to Consadole in recent years The sheer presence of a figure as influential and respected as Ono means his value -- in recent times -- has come off the field rather than on it During his spell with Consadole, Thailand star Chanathip Songkrasin recounted how Ono was still one of the most technically gifted players in training suggests he could even handle the physical demands of playing on even for another season or two But as has been the case throughout his career injuries have not been kind to Ono and they certainly taken their toll Shinji Ono tasted success during his time in Europe when he won the 2001-02 UEFA Cup with Feyenoord alongside a then-18-year-old Robin van Persie as well as stalwarts such as Pierre van Hooijdonk and Jon Dahl Tomasson. Tim De Waele/Getty ImagesYet as his career came to an end on the weekend amid little fanfare outside of Japan it can be guaranteed that his brilliance will live on in the memories of those who had the pleasure of watching him in the past His raw talent saw him become the youngest player in the Japan squad for their FIFA World Cup debut in 1998 at only 18 After starring consistenly for Urawa, Europe soon came calling in the form of Eredivisie giants Feyenoord and it did not take long for him to establish himself as a important first-team player -- winning the now-defunct UEFA Cup along the way in 2002 Equally adept on either foot with a glorious passing range but with his greatest weapon arguably his vision to see the play before it unfolded Ono was at his best when he was allowed the freedom to roam wherever he wished on the pitch Netherlands legend Wesley Sneijder was once quoted identifying Ono as his toughest direct opponent while his Japanese nickname (天才 or tensai) simply means "genius" Even in his later years, when he made Australia's A-League his final overseas stop, it did not take long for him to be adored by the Western Sydney Wanderers faithful -- especially after a world-class brace that inspired a win over Melbourne Victory The first saw him effortlessly control a looping clearance while moving away from goal before throwing in a a casual juggle that got him facing the right direction and then clinically firing through the legs of a defender and into the bottom corner all without the ball ever touching the ground once far less complicated but arguably more exquisite saw him receive possession on the edge of the area steady himself as he took a half-second to assess what his best option was before proceeding to bend a sublime curling effort inside the post And when considering that Ono usually preferred to lay on an assist rather than go for goal despite his penchant for the spectacular it perhaps begs the question just how much more iconic a player he might have been had he just been slightly less selfless he may not be among the first names that spring to mind in a conversation around the best players Asian football has produced But as he goes quietly into the night -- which perhaps might just be the way he would have preferred it to be -- the genius of Ono will linger on in the memory of those who witnessed it over the past 25 years Sibbinda -Richwell Mazumo kwatokora kuruganesa udivi wendi wonondima yipo atameke unandima woposiruwoasi ngesefa Age kurandesa oyo ana zangura konkarapamwe zomoSibbinda Age kwapura asi eyi yokuninka asi nkenye eyi ono hara ngano vayikupe yahaga momawiza moomu tupu ngayigeve mwene sankendengere nomungavagwana nomboroto dokutunda meguru valye vaIsarael ngavagwana manna zokutunda meguru zigwire mwaza sirongo Ose twahepa nye kundindira epangrero litupe nayinye tuna hepa makura turuganeni yipo tuparuke,’’ yimo ana kutanta ngoso age kuna sikama pokatji kosipata sendi esi sina kupekema Age kwatameka nosikunino sendi esi kutunda mo 2022 nomusunda gumwe ngoso gevhu Nampili ngomu yakara asi nondima kwakara nomaudigu gawo age nkenye apa kupinduka monongura ponovili 03h00 akarugane mosipata sendi komeho aka ze koyirugana yendi yourongi age tayalipakerere kumwe nepata lyendi vayatwikire kuyalima ndi kupakera sinka sikunino sawo esi gava sigira guhyawo age kugava nye kovatungimo ntani kutwara kodoropa zaKatima Mulilo ame kukuna yikwa hidi eyi nokurandesa konkarapamwe kondando zongwa,’’ yimo ana kutanta Age kurandesa sprnch pokatji koN$5 no N$10 ngoso kulisiga tupu asi unene ngapi lina kara.  Mazumo age simpe kuna kwakara nonondima omu gaweka nongombe yilya ntani katjama ntani hena sweet potatoes pontambo zonene age simpe kuna kutanta asi kuna kurugana nawa morwa age galituramo unene ntani kurugana unene.  ‘’ Nampili asi sinzi kukondja unene age gatompora mokuzangura nonsako ntambali depungu Yina kara asi yina gwanene kwendi yina kara asi ezi mvhura zodigu Age nare gaturapo nokuzogera nonkarapamwe ngano vayamborore ngesi vamana nare zina kara asi zokorutjeno age kuruganesa yihemere yiyo ayininkisa alivinduke atekere Age ngesi kuna kuhundira kovasamaritani wonkenda vavavatereko mema neyi yokutekeresa ntani darate vamange kevango lyawo makura awo kuna kutjira asi kuvhura kuzonagura nomulihu noyimeno yawo sikunino sange hawe ngano tasi wapa unene.  Mokugwedako, age kunakumona asi nsene agwana mbatero ngano tamborora ekero lyamwene yipo agwederere sipata sendi, nokugwedako nye morupe ronondja mosirongo. Age kuna kugava mukumo kovadinkantu asi vahayingira tupu, nye vahepa kurugana yonondima yipo asi vahandindira tupu epangero asi yiylo nalivavatera. -anakale@nepc.com.na About Us Vacancies Procurement Gallery Contact Us We use cookies to enhance your browsing experience We use cookies to help you navigate efficiently and perform certain functions You will find detailed information about all cookies under each consent category below The cookies that are categorized as "Necessary" are stored on your browser as they are essential for enabling the basic functionalities of the site We also use third-party cookies that help us analyze how you use this website and provide the content and advertisements that are relevant to you These cookies will only be stored in your browser with your prior consent You can choose to enable or disable some or all of these cookies but disabling some of them may affect your browsing experience Necessary cookies are required to enable the basic features of this site such as providing secure log-in or adjusting your consent preferences These cookies do not store any personally identifiable data Functional cookies help perform certain functionalities like sharing the content of the website on social media platforms Analytical cookies are used to understand how visitors interact with the website These cookies help provide information on metrics such as the number of visitors Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors Advertisement cookies are used to provide visitors with customized advertisements based on the pages you visited previously and to analyze the effectiveness of the ad campaigns Please enable JS and disable any ad blocker Metrics details The study aims to identify histological classifiers from histopathological images of oral squamous cell carcinoma using convolutional neural network (CNN) deep learning models and shows how the results can improve diagnosis Histopathological samples of oral squamous cell carcinoma were prepared by oral pathologists Images were divided into tiles on a virtual slide VGG16 and ResNet50 with the optimizers stochastic gradient descent with momentum and spectral angle mapper (SAM) were used with and without a learning rate scheduler The conditions for achieving good CNN performances were identified by examining performance metrics We used ROCAUC to statistically evaluate diagnostic performance improvement of six oral pathologists using the results from the selected CNN model for assisted diagnosis VGG16 with SAM showed the best performance The diagnostic performances of the oral pathologists statistically significantly improved when the diagnostic results of the deep learning model were used as supplementary diagnoses (p-value = 0.031) By considering the learning results of deep learning model classifiers the diagnostic accuracy of pathologists can be improved This study contributes to the application of highly reliable deep learning models for oral pathological diagnosis the importance and workload of pathologists in diagnosing this disease are increasing Pathologists must make many histological diagnoses and large amounts of experience and learning are required to achieve an accurate diagnosis double-checking is a useful technique in histopathological diagnoses and has been adopted in clinical practice we hypothesize that the use of deep learning may contribute to improving the accuracy of histopathological diagnoses The primary purpose of this study is to identify an effective histological classifier from histopathological images of oral squamous cell carcinoma using a deep learning CNN model and then to clarify the classification of the performance of the classifier The second purpose is to show whether the learning results of the identified effective deep learning classifier model can contribute to improving the diagnostic performance of oral pathologists Table 1 shows the results of the performance metrics obtained with and without a learning rate scheduler for the SGDM and SAM optimizers on VGG16 and ReaNet50 With the introduction of learning rate scheduling SGDM exhibited improved performance metrics except for the area under the curve (AUC) VGG16 had higher performance metrics under all conditions and ResNet50 had higher performance metrics for all conditions except for AUC when SAM was used VGG16 with SAM showed the highest performance the best deep learning model was found to be VGG16 with SAM as the optimizer Table 2 shows the AUC the highest AUC without an assistive diagnosis was for oral pathologist #4 who obtained a macro average of 0.95 (95% confidence interval; 0.942–0.950) and a micro average of 0.95 (95% confidence interval; 0.946–0.955) the macro average was 0.98 (95% confidence interval; 0.976–0.980) and the micro average was 0.95 (95% confidence interval; 0.976–0.982) Oral pathologist #1 was most effective when an assistive diagnosis was used A macro mean of 0.80 (95% confidence interval; 0.795–0.810) and a micro mean of 0.79 (95% confidence interval; 0.776–0.791) were obtained without an assistive diagnosis When the assistive diagnosis component was used the macro average was 0.97 (95% confidence interval; 0.960–0.966) and the micro average was 0.97 (95% confidence interval; 0.958–0.965) The diagnostic performances of all pathologists were improved in terms of the AUC using the assistive diagnosis technique Comparison of oral pathologists' diagnoses with and without deep learning assistance considering the ROC curve using macro mean values. Comparison of oral pathologists' diagnoses with and without deep learning assistance considering the ROC curve using micro mean values Statistical comparison of the oral pathologist's diagnoses with and without deep learning assistance This study demonstrated that the most effective classification model for classifying histopathological images of oral squamous cell carcinoma using deep learning uses VGG16 with a learning rate scheduler and the SAM optimizer Diagnoses using deep-learning assistance were shown to contribute to the improvement of the diagnostic accuracy of oral pathologists by considering the learning results of the classifier that were obtained using the best model Most studies have divided oral squamous cell carcinoma into normal tissue or benign and malignant tumors we targeted all cropped images that contained cells the proposed CNN model achieved a high classification diagnostic performance for the multiclass classification of complex datasets have provided both correct and incorrect evaluations we evaluated macro- and micro-averaged AUC techniques using continuous confidence and this is the first study to evaluate the effectiveness of deep-learning-assisted diagnoses in oral histopathology Effect sizes may be used to determine the number of observers that will be present in future similar studies The results of this study may provide a basis for the application of reliable deep learning methods in histopathological diagnoses and many other optimizers and learning rate schedulers were not investigated To verify the use of more complex CNN models sufficient resources that can withstand the required computational costs are needed the pathological tissue images were verified at only one facility and the verification of external validity using external data is also required to confirm the effectiveness of more robust auxiliary deep learning diagnosis methods dataset-splitting techniques can affect the generalizability of deep learning techniques and divided the training data into test data from those images comparing the evaluation methods for the learning and test data for each histopathological specimen will be required in future studies to evaluate the effectiveness of deep learning assistance we first made a diagnosis without using deep learning and then made a diagnosis using deep learning assistance The interval between evaluations varied according to the pathologist who performed each evaluation The same test sample may affect the pathologist's subjective judgment; therefore considering evaluations after a long period we identified an effective histological classifier from histopathological images of oral squamous cell carcinoma and clarified the classification performance of this classifier using deep learning with a learning rate scheduler and SAM optimizer This system was statistically demonstrated to improve the diagnostic accuracy of pathologists by referring to the learning results of the classifiers that have undergone deep learning This study provides a basis for applying reliable deep learning systems in the field of oral pathology diagnosis Overall flow of the research on deep learning classification models for oral histopathology This study was approved by the Institutional Review Board (IRB) of the Kagawa Prefectural Central Hospital Ethics Committee (the Institutional Review Boards of Kagawa Prefectural Central Hospital which is a non-interventional retrospective study design It is an analytical study with fully anonymized data and the need for informed consent was waived Because the data were evaluated retrospectively and were solely obtained for treatment purposes a requirement of informed consent was waived by the IRB of the Kagawa Prefectural Central Hospital Ethics Committee written and verbal informed consent was not obtained from the patients from whom pathological specimens were obtained This research uses existing sample information and obtaining direct informed consent from all research subjects is difficult at the request of research subjects or their representatives directly to the hospital ethics committee informed consent was denied by timed opportunities to refuse participation when requested to use specimen information that could identify research subjects or to provide it to other research institutions This study was conducted in accordance with the Declaration of Helsinki and according to the rules and protocol approved by the IRB The dataset used slide glasses of five biopsy specimens stained with hematoxylin and eosin (H & E) The five specimens were three cases of tongue cancer and two cases of oral floor cancer [four cases for men one case for women; average age: 73 years (47 to 90 years)] The glass slides were scanned with an Aperio AT2 scanner (Leica Biosystems Illinois) at 40-times magnification to create a whole slide image (WSI) The created WSI was tiled using OpenSlide (version 3.4.1 The cropped images were output in portable network graphics (PNG) format at 256-by-256 pixels including cells with an oval nuclear shape and abnormal mitotic figures inconspicuous nucleoli; (2) squamous cell carcinoma including cells with an irregular nuclear shape and 5762 other) were professionally labeled and conversion (30% up/down/left/right) were performed randomly and the missing part of the image was complemented using the reflection method the dataset was split into separate training and testing datasets at a ratio of 90:10 the validation data consisted of 10% of the training data The model performance evaluation used the average of the analysis results for each fold to obtain the results for the entire dataset the cross-entropy obtained from the following equation was used: ti is the true label; yi is the predicted probability of class i We chose stochastic gradient descent with momentum (SGDM) and sharpness aware minimization (SAM) as the optimization algorithms for this study SGDM is expressed by the following formula: wt is the parameter; η is the learning rate; ∇L (w) is the differentiation with parameters of the loss function; α is the momentum S is the set of data; w is the parameter; λ is the L2 regularization coefficient; Ls is the loss function; ρ is the neighborhood size All deep learning analyses were performed using a 64 bit Ubuntu 18.04.5 LTS operating system (Canonical Ltd. UK) and NVIDIA GeForce Tesla V100-SXM2 16 GB graphics processing unit (NVIDIA The process of deep learning classification was implemented using Keras (version.2.7.0) All CNN models were trained at 300 epochs and 32 mini-batch sizes and did not use premature termination These deep learning analysis processes were repeated 30 times for each model and different random seeds were used for each model All deep learning models were evaluated in terms of their accuracy, precision, recall, specificity, F1 score, and AUC calculated from ROC as performance metrics. More information on each performance metric can be found in Appendix S1 Six oral pathologists participated in this study—three board-certified specialists in oral pathology and three specialists in oral pathology who have not yet been board-certified Each oral pathologist was informed about the composition of the images (normal and squamous cell carcinoma) and they reviewed the images individually to make diagnoses diagnoses were made without the deep learning assistance and later deep learning assistance was used in the diagnoses The correct diagnosis for each image was not communicated to the oral pathologist evaluators until after the two tests were completed The pathologists performed the tests individually and promised not to share their results with the other observers The diagnostic method used was the continuous confidence method in which scores were given on a free scale according to various criteria The method used a visual scale from 0 to 100 to determine the certainty of normal the SoftMax function was used to convert the total output values of the three categories to 1.0 (100%) we analyzed the effectiveness of deep-learning-assisted diagnosis using the ROC curve and ROCAUC for aiding the diagnoses of oral pathologists Using macro- and micro-average values of the results we compared the effect of deep-learning-assisted diagnosis using ROC and evaluated the effect of using deep learning on the diagnostic performances of oral pathologists The effect size is a metric that was proposed by Cohen that is determined based on the criteria proposed by Sawilloski28 The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries A deep-learning pipeline for the diagnosis and discrimination of viral non-viral and COVID-19 pneumonia from chest X-ray images Deep neural networks for dental implant system classification Evaluation of multi-task learning in deep learning-based positioning classification of mandibular third molars Deep learning enables automatic classification of emphysema pattern at CT Emerging role of deep learning-based artificial intelligence in tumor pathology Pathologists can get it right the first time Sharpness-Aware Minimization for Efficiently Improving Generalization Effective deep learning for oral exfoliative cytology classification Deep learning-based total kidney volume segmentation in autosomal dominant polycystic kidney disease using attention Using adversarial images to assess the robustness of deep learning models trained on diagnostic images in oncology Histopathologic oral cancer prediction using oral squamous cell carcinoma biopsy empowered with transfer learning Deep learning on oral squamous cell carcinoma ex vivo fluorescent confocal microscopy data: A feasibility study Automated detection and classification of oral lesions using deep learning for early detection of oral cancer Musculoskeletal radiologist-level performance by using deep learning for detection of scaphoid fractures on conventional multi-view radiographs of hand and wrist Deep learning-enabled pelvic ultrasound images for accurate diagnosis of ovarian cancer in China: A retrospective Artificial intelligence improves the accuracy in histologic classification of breast lesions Sample size determination and power analysis using the G*Power software Nuclear features in oral squamous cell carcinoma: A computer-assisted microscopic study Simonyan, K. & Zisserman, A. Very deep convolutional networks for large-scale image recognition. arXiv https://doi.org/10.48550/arxiv.1409.1556 (2014) He, K., Zhang, X., Ren, S. & Sun, J. Deep residual learning for image recognition. arXiv https://doi.org/10.48550/arxiv.1512.03385 (2015) A study of cross-validation and bootstrap for accuracy estimation and model selection An Improved Analysis of Stochastic Gradient Descent with Momentum (2020) confidence interval and statistical significance: A practical guide for biologists Download references This work was indirectly supported by JSPS KAKENHI (Grant Number JP19K19158) and JST Department of Oral and Maxillofacial Surgery Center for Healthcare Information Technology Tokai National Higher Education and Research System All authors analyzed and interpreted the data All authors have read and approved the published version of the manuscript The 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/s41598-023-38343-y Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research a trip to Japan offers the opportunity to observe rich culture For one DeKalb County School District (DCSD) teacher a trip to Japan offered the chance to reconsider how she approaches education Fernbank Elementary English as a Second Language (ESOL) teacher LaShia Brooks was one of 31 educators to visit Japan as part of an international partnership with the Japanese Chamber of Commerce The purpose of the trip—which lasted from June 24 until July 5—was to foster a deeper understanding of Japanese culture and native Japanese students studying outside of Japan “It’s important to be able to connect with ESOL students through real-world experiences,” Brooks said I can pick up on cultural norms and connect on a deeper level Even if it’s just mentioning a popular snack or place It’s letting them know you’ve been on their turf You would be surprised how far that can get you.” Brooks was invited on the trip after being nominated by a former Fernbank Elementary student Following an application process and a three-person panel interview Brooks was invited to explore the bustling and the culturally resonant history of Kyoto – all expenses paid “Kota cried, ‘Yes!’” Brooks said. “I cried after realizing how much he wanted this for me.” who had only traveled abroad to the Caribbean previous to the trip said she felt a mix of excitement and anxiety Brooks brushed up on her Japanese via an old Rosetta Stone course—asking Kota for help when she needed it Fortunately Brooks found all of her anxieties to be unfounded “I have never experienced anything like that in my entire life.” Brooks’ trip included visits to elementary and entertainment venues to take in the entire spectrum of the country The contrast of the paces between cities with the countryside the amount of historical weight at both cities and smaller towns and the general order of it all struck Brooks intellectually and emotionally One of the stark differences Brooks noticed was the country’s approach to generally everything Japanese society tends to take a communal perspective Public spaces are treated with great respect Solving a problem for one person generally includes all involved “People in a public space were very considerate of their volume and personal space,” Brooks said Even in the crowd there seemed to be organized I definitely felt like a part of ‘it.’” and when one student has a hard time understanding the entire group contributes until he or she grasps the concept With such great respect for the learning process comes greater respect for education as a whole Students and teachers commit to one extra day per week for school and some even start their day before 5 a.m As a first time visitor and longtime educator Brooks was surprised with how well-received the group was for merely being teachers “School culture is considered more worthwhile—they enjoy it,” Brooks said “Everything is taken with greater pride,” Brooks said “We were treated first class no matter where we went Brooks gained a new sense of consideration for ESOL students When comparing Japanese culture with American culture she said she could see how moving stateside could be overwhelming for certain students Her only wish is that this program existed throughout the world in almost every country this consideration may have been the ultimate lesson taught by the life-changing trip “I think each educator was meant to determine their own purpose,” Brooks said “It makes me consider more for my students It has given me pause in my approach to things and the country wants to better serve their own people abroad.” View pictures from the Japan trip DeKalb County School District does not discriminate on the basis of race, color, national origin, sex, disability, genetic information or age in its programs and activities, and provides equal access to the Boys Scouts and other designated youth groups. The following person has been designated to handle inquiries regarding the non-discrimination policies including Title IX: Marissa Key, Executive Director - Employee Relations| P. 678.676.0503 © 2003 - 2022 DeKalb County Board of Education | 1701 Mountain Industrial Boulevard · Stone Mountain, GA 30083 | P: 678.676.1200 We are creating technology to overcome this final “cliff” in the development of atomic frequency standards and bring them to a size and price-point comparable to modern microprocessors. As communication networks evolve from mere telephony to including diverse applications like automobiles, robots or aerial drones, precise timing sources at the endpoint will help provide the required efficiency, security and stability for future applications. Future applications will depend on such devices to maintain authenticated time and position even when the signals of global navigational satellite systems are unavailable. 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Volume 6 - 2015 | https://doi.org/10.3389/fpls.2015.00890 This article is part of the Research TopicSeasonality of woody plants in cold climates: adaptations, regulation and modelingView all 24 articles Boreal coniferous species with wide geographic distributions show substantial variation in autumn cold acclimation among populations To determine how this variation is inherited across generations we conducted a progeny test and examined the development of cold hardening in open-pollinated second-generation (F2) progeny of Abies sachalinensis The F1 parents had different genetic backgrounds resulting from reciprocal interpopulational crosses between low-elevation (L) and high-elevation (H) populations: L × L Paternity analysis of the F2 progeny using molecular genetic markers showed that 91.3% of the fathers were located in surrounding stands of the F1 planting site (i.e. The remaining fathers were assigned to F1 parents of the L × L cross-type This indicates that the high-elevation genome in the F1 parents was not inherited by the F2 population via pollen flow The timing of autumn cold acclimation in the F2 progeny depended on the cross-type of the F1 mother The progeny of H × H mothers showed less damage in freezing tests than the progeny of other cross-types Statistical modeling supported a linear effect of genome origin variation in freezing damage was explained by the proportion of maternally inherited high-elevation genome These results suggest that autumn cold acclimation was partly explained by the additive effect of the responsible maternal genome the offspring that inherited a greater proportion of the high-elevation genome developed cold hardiness earlier Genome-based variation in the regulation of autumn cold acclimation matched the local climatic conditions which may be a key factor in elevation-dependent adaptation Controlled crosses using mother plants that have been exposed to the same environmental conditions can be used to test the effects of genome inheritance on the regulation of the autumn cold acclimation previous ecological studies have not extensively evaluated the complex effects of genome inheritance on the development of cold acclimation in trees A map of the study site (A) and the experimental design using F2 progeny of interpopulational crosses of Abies sachalinensis (B) Two natural populations used for crossing were located where the latitudinal and longitudinal lines cross in panel (A) A planting site of the F1 population and a common garden trial for the F2 progeny were established in the same area The F1 population was produced using reciprocal crossing between low-elevation (L) and high-elevation (H) populations in 1979 Open-pollinated seeds were collected from the F1 mother trees in 2009 The resulting F2 progeny had various genetic backgrounds resulting from the maternal cross-type we used open pollination to obtain second-generation (F2) progeny for testing the genetic basis of variation in phenological traits The maternal population (F1) was produced by reciprocal crosses between two distinct ecotypes: a population inhabiting low elevations and a population inhabiting high elevations The genetic background of the maternal F1 trees varied among cross-types (low × low and the trees were planted at a single site in order to ensure their exposure to the same environment we could evaluate the effects of genome inheritance by examining the traits of the F2 population we conducted (1) paternity analysis based on microsatellite markers and (2) statistical modeling of freezing damage Our objectives were to (1) measure variation in the timing of autumn cold acclimation of the F2 progeny and (2) establish whether this variation could be explained by the genomic background inherited from the low-elevation or high-elevation populations the primary factor changing with elevation is air temperature Based on monitoring data recorded hourly from 2009 to 2010 at 230 m the annual and winter (October–March) mean temperatures were 6.53°C and -1.78°C Temperatures at 1100 m were 1.75°C and -6.43°C Collected seeds were stored at -3°C until use Number of F1 trees of Abies sachalinensis that were living or used to collect open-pollinated seeds in 2009 the cross-type differences of the mother of the F2 progeny were reflected in some functional traits such as the germination rates and 2-year heights Germination rates and 2-year heights of F2 progeny in a common garden trial at 230 m a.s.l To estimate the genome composition of the F2 progeny, we performed molecular genetic analysis using nuclear microsatellite (nSSR) and chloroplast microsatellite (cpSSR) markers. In the present study, we used four nSSRs: As08, As16, As32 (Lian et al., 2007), and NFH 15 (Hansen et al., 2005), and three cpSSRs: pt30204, pt71936 (Vendramin et al., 1996), and pt30249 (Liepelt et al., 2001) Samples selected for SSR genotyping consisted of the 22 flowering F1 trees described in Table 1 plus 80 of their F2 progeny. These 80 F2 progeny were derived from seeds collected in 2009, and were composed of 16 progeny from each of five F1 trees selected across the cross-types (Table 3) These seedlings were germinated in an indoor growth chamber Genetic diversity statistics of the F1 trees that flowered in 2009 and their open-pollinated F2 progeny We considered the 22 flowering F1 trees as candidate pollen parents of the F2 progeny because no other trees were flowering at the F1 planting site trees outside of the F1 population may have also served as pollen parents because the F1 planting site was surrounded by mature artificial forests The origin of these artificial forests was a local lowland forest (200–300 m) that was different from the forests used to create the F1 population We set the genotyping error to 1% with an 80% confidence level for the 10000 cycle-simulations for the paternity assignment After assigning fathers using CERVUS, we performed a simple exclusion procedure using three cpSSRs. The F2 progeny and the assumed F1 pollen parents were required to have matching cpSSR haplotypes (Abies cpSSRs are paternally inherited; Vendramin and Ziegenhagen, 1997) We assumed that CERVUS assigned the correct pollen parent when the genotypes of the F2 progeny and assigned pollen parent matched for all three cpSSR markers (considering genotyping errors) we assumed the pollen came from outside of the F1 planting site Combining the results from CERVUS and the exclusion procedure we estimated the proportion of selfed seeds and seeds sired by fathers outside the studied F1 population We also estimated the composition of the F1 trees among four cross-types Freezing damage was scored using six classes of needle discoloration (brown needles) as follows: 0 (no damage) 1 (1–20% of needles were discolored) we used 10 F2 seedlings from each F1 mother tree For some maternal trees with fewer seedlings 605 seedlings were used in the freezing test (121 seedlings × 5 test conditions) Because no freezing damage was observed in the -15°C treatment in Tests 2 and 3 we excluded these data from subsequent analysis three test conditions were defined: T1 involved freezing at -15°C in Test 1 T2 involved freezing at -30°C in Test 2 and T3 involved freezing at -30°C in Test 3 we used a nested ANOVA to study the effect of maternal cross-type and the effect of mother trees on freezing damage in the F2 progeny where Yijk is the freezing damage score of the k-th progeny (k; 1-10) of the j-th mother tree (j; 1-4) in the i-th cross-type (i; 1-4) Mj (Ci) is the effect of the j-th mother tree nested in the i-th cross-type We also evaluated the effect of low-elevation and high-elevation plant genomes on freezing damage using the following statistical model that includes all test conditions We assumed that freezing damage in the F2 progeny is affected by the genetic inheritance we constructed the full model with all assumed effects We then assessed which types of variables were appropriate for the effect of the plant genome (C) to describe the freezing damage score we used the following four types of the genetic effects as C in Models 2–5: Model 2: Linear effect (0 for L × L; 0.25 for L × H and H × L; 0.5 for H × H) Model 3: Categorical effect (L × L; L × H; H × L; H × H) Model 4: Categorical interaction effect (“NoHyb” for L × L and H × H; “HybLH” for L × H; “HybHL” for H × L) Model 5: Linear effect + interaction effect (0 for L × L; 0.25 + α for L × H; 0.25 - α for H × L; 0.5 for H × H) the proportion of high-elevation genome inherited from the maternal parent was used Numeric values indicating the linear effect of the maternal genome origin were assigned to C The highest value was assigned to the H × H cross-type was assigned to the L × L cross-type Model 2 should show a good fit if freezing damage was closely related to the amount of high-elevation genome inherited from the maternal trees (i.e. C was set to one of four categorical values indicating the cross-type of the maternal trees Model 3 should fit well if the maternal origin is important but the proportion of high-elevation genome is not Model 4 included a genome interaction effect C was set to one of three categorical values: NoHyb we included both the cross-type linear effect and genome interaction effect C was partitioned into the combination of the variables used in Models 2 and 4 their observed heterozygosity (HO) was higher in the F2 progeny than in the F1 trees resulting in a negative fixation index (FIS) Paternity analysis using four nSSR markers showed that 12 F2 progeny had a father from the candidate F1 pollen parent group seven F2 progeny out of 12 were not excluded the cross-type of all of the assigned F1 fathers was L × L There were no progeny with genotypes composed solely of maternal alleles most of the F2 progeny had novel alleles in comparison with the candidate F1 pollen donors Freezing damage of F2 progeny after a freezing test at -15°C in Test 1 (A) Three freezing tests were conducted in autumn at a 2-week interval The freezing damage scores range from 5 (complete needle discoloration) to 0 (no discoloration) Observed frequencies were shown by arraying the cross-type of each mother (on the lower x-axis) The maximum scale of the number of observations (on the top x-axis) was set to 18 for each of the four cross-types in Tests 1 and 2 and set to 30 for each of the four cross-types in Test 3 The ANOVA indicated a significant difference among mother trees within cross-types [M (C)] for treatment T1 (Table 4). For treatment T2, a significant difference was detected among maternal cross-types (C), but not among mother trees [M (C)]. For treatment T3, however, statistical analysis was not possible due to limited variation in freezing damage score, as shown in Figure 2 ANOVA of freezing damage in the F2 progeny for each test condition A significant negative effect was observed for C indicating that the freezing damage tended to be lower in the progeny that inherited a higher proportion of the high-elevation genome Candidate model components and Akaike Information Criterion (AIC) values after model selection the best model among all candidate models (Models 2–5) for predicting freezing damage in the F2 progeny (standard errors are in parentheses) our evaluation method is sufficient for detecting phenotypic variation associated with cold acclimation in this species we further evaluated the effects of genetic background of the trees using molecular markers and statistical models to improve our understanding of the local adaptation and the evolution of autumn cold acclimation The combined use of nSSR and cpSSR markers made it possible to assign pollen parents to A. sachalinensis seedlings. We assigned 22 candidate F1 pollen parents to the F2 progeny with consideration for genotyping error and assignment confidence (Slavov et al., 2005) We assumed that cryptic gene flow had little effect because the genetic origin of the F1 trees was different from that of the artificial forests surrounding the F1 planting site when the paternity analysis found no possible fathers among the candidates we assumed that the pollen parent was outside of the F1 planting site and presumably derived from the surrounding mature plantations the amount of pollen from the F1 planting site must be lower than that from the surrounding mature plantations the results obtained by paternity analysis are reasonable it is not surprising that outside pollen flow was predominant at our F1 planting site The negative FIS in F2 indicated that the chances of mating between an F1 mother and its relatives were low These results support our conclusions concerning pollen flow from outside the F1 planting site We concluded that only a few F1 fathers contributed to the F2 progeny because of the high levels of pollen flow from the surrounding mature plantations the F1 trees that were assigned as fathers (seven progeny) were all derived from the L × L cross-type This indicates that the high-elevation plant genome from the F1 population was not inherited by the F2 population to any great degree Our modeling analysis revealed that the regulation of autumn cold acclimation could be well explained by the linear effect of the genome origin. The model using the expected proportion of high-elevation genome as a fixed effect was selected as the best model (Table 5) variation in autumn cold acclimation was explained by variation in the maternal genome composition The F2 progeny that inherited a greater proportion of the high-elevation genome showed earlier cold acclimation In comparison with models considering such effects the model including only the effect of genome origin showed the best fit This result could be partly explained by the fact that all maternal trees were exposed to the same environmental conditions in the absence of a specific cross-type effect it would be reasonable to expect that the autumn cold acclimation of A sachalinensis would show a linear pattern based on the proportion of low-elevation and high-elevation plant genomes The genome-based control of phenological traits may be the mechanism responsible for the elevation-dependent adaptation of this species it may be difficult to track rapid climate change because of the mismatch of locally adapted autumn phenology the presence of adaptive genes and their potential for climatic adaptation have not been sufficiently examined Future studies may be needed to evaluate this subject in detail by applying powerful molecular techniques such as the analysis of quantitative trait loci and/or genome scanning The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The authors are deeply grateful to Glenn Howe for critical comments on the manuscript The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00890 “Genecology and gene resource management strategies for conifer cold hardiness,” in Conifer Cold Hardiness Columbo (Dordrecht: Kluwer Academic Publishers) Google Scholar migration or extirpation: climate change outcomes for tree populations Cané-Retamales Bayesian threshold analysis of breeding values genetic correlation and heritability of flowering intensity in Eucalyptus cladocalyx under arid conditions Christensen, R. 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Front. Plant Sci. 6:890. doi: 10.3389/fpls.2015.00890 Copyright © 2015 Ishizuka, Ono, Hara and Goto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Wataru Ishizuka, d2F0YXJ1LmlzaGlAZ21haWwuY29t In 2018 Japanese artist Setsuko Ono will bring her work to London for the very first time with two solo exhibitions taking place at The Daiwa Anglo-Japanese Foundation in February and Asia House in March 2018 Setsuko was born in Tokyo and grew up between Japan Setsuko worked at the World Bank for 28 years while pursuing a formal art education in Washington but she only began exhibiting her art once she retired in 2003 Setsuko had her first exhibition at the Eighth Havana Biennial Setsuko has had 14 permanent public sculptures installed in Havana The London exhibitions will include both sculpture and mixed media paintings and visitors will be able to view Setsuko’s permanent installations at Hara Museum in central Tokyo Setsuko creates steel sculptures characterised by their cut-out shapes forming opened and closed figures and designs that integrate into the outdoors The cut-out silhouettes are bent in an animated way while the cut out negative lets the sunlight and views of nature through cut out sculptures are created from sheets of steel Inspired by meeting her musical hero John Cage as a teenager detailed blueprints or preliminary drawings Setsuko’s recent work includes mixed media paintings that reflect her interest in international politics I have read and agree to the terms & conditions Contact us: info@theartsshelf.com HOMEABOUT USMEET THE TEAMPRIVACY POLICYADVERTISING INFOCONTACT Metrics details Glycoprotein nonmetastatic melanoma protein B (GPNMB) plays important roles in various types of cancer and amyotrophic lateral sclerosis (ALS) The details of GPNMB function and its interacting protein have not been clarified to identify GPNMB binding partners on the cell membrane we used membrane protein library/BLOTCHIP-MS technology which enables us to analyze all cell membrane proteins as binding partners of the GPNMB extracellular fragment we identified the alpha subunits of Na+/K+-ATPase (NKA) as a possible binding partner We confirmed the interaction between the GPNMB extracellular fragment and NKA by immunoprecipitation and immunostaining in NSC-34 cells endogenous GPNMB extracellular fragment bound to and colocalized with NKA alpha subunits also bound to and colocalized with NKA alpha subunits we found that the GPNMB extracellular fragment had neuroprotective effects and activated the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathways via NKA These findings indicated that NKA may act as a novel “receptor” for the GPNMB extracellular fragment offering additional molecular targets for the treatment of GPNMB-related diseases we hypothesized that the extracellular fragment of GPNMB binds a receptor or proteins on the plasma membrane and then activates the PI3K/Akt and MEK/ERK pathways we have identified the Na+/K+-ATPase as a receptor for the extracellular fragment of GPNMB that mediates activation of cellular signaling pathways and subsequent neuroprotective effects To identify novel interacting proteins of GPNMB we conducted a comprehensive search using Membrane Protein Library (MPL)/BLOTCHIP-MS technology MPL enables users to extract all cellular membrane proteins and reconstitute them in artificial lipid bilayers composed of phospholipid liposomes to protein function By differential analysis of SDS-PAGE electrophoresis profile the MPLs and 120 kDa and were considered possible receptors (A) An equal amount of cell lysate was subjected immunoprecipitation (IP) by anti-NKA α1 antibody and detected by western blot using anti-GPNMB antibody (B) Co-immunostaining was conducted to localize NAK and GPNMB by anti-GPNMB antibody (red: a and d) and anti-NAK α1 antibody (green: b) (C) 661 W cells were plated at 2 × 105cells/well in 6-well plates and incubated overnight The cell lysate was subjected immunoprecipitation (IP) by anti-NKA α1 antibody (D) 661 W cells were seeded at 5 × 104cells/well in a Lab-Tek II Chamber slide (Thermo Fisher Scientific) and incubated overnight Co-immunostaining in 661 W cell was conducted to localize NAK and GPNMB by anti-GPNMB antibody (red: a and d) and anti-NAK α1antibody (green: b) (E) NSC-34 cells were pretreated with human recombinant GPNMB and subjected to IP by anti-NAK α1 antibody and detected by western blot with anti-human IgG antibody (F) Co-immunostaining was conducted to localize of human recombinant GPNMB (green: a The cells were pretreated with negative-control siRNA The cells were pretreated with NAK α1 siRNA and then treated with the human recombinant GPNMB (c To determine the specificity of the interaction of GPNMB with NKA, cells were pretreated with NKA α1 small-interfering RNA (siRNA) or negative-control siRNA. The human recombinant GPNMB partially colocalized with NKA α1 on the plasma membrane of cells transfected with negative-control siRNA (Fig. 2F) the human recombinant GPNMB localized not only on the cell membrane the fluorescence was attenuated by NKA α1 siRNA transfection these results suggested that the endogenous and exogenous GPNMB extracellular fragments interact with NKA alpha subunits NSC-34 cells were loaded with the membrane potential dye The fluorescence signal of HLB021-152 was measured using microplate reader before 5 and 60 min after adding human recombinant GPNMB (F0) or ouabain (F) (A) NSC-34 cells were treated with the human recombinant GPNMB (0.025 Each column and bar represents the mean ± S.E.M (B) NSC-34 cells were treated with human recombinant GPNMB (2.5 μg/ml) or ouabain Next, to confirm that the effects of GPNMB on the cellular membrane potential were mediated by NKA, we investigated whether the change in cellular membrane potential was blocked by the NKA inhibitor ouabain. The intensity of fluorescence increased initially after stimulation with 2.5 μg/ml human recombinant GPNMB; however, this increase was blocked by ouabain (Fig. 3B) This suggests that the extracellular fragment of GPNMB may activate NKA by binding to NKA Cells were treated with vehicle or with human recombinant GPNMB (2.5 μg/ml) (A) ouabain (10 μM) and (B) sanguinarine (0.3 μM) The number of cells displaying PI or Hoechst 33342 fluorescence was counted and the cell death rate was expressed as the ratio of PI-positive cells to Hoechst 33342-positive cells NSC34 cells were treated with human recombinant GPNMB (2.5 μg/ml) and ouabain (10 μM) or sanguinarine (0.3 μM) (A) The level of phosphorylated Akt was quantified relative to total Akt (B) The level of phosphorylated ERK1/2 was quantified relative to total ERK (C) The level of p-Src protein was examined at different time points (0–60 min) after incubating with the recombinant extracellular GPNMB (2.5 μg/ml) The level of phosphorylated Src was quantified relative to total Src Statistical comparisons were made using Dunnett’s test (vs (A) Cells were transfected with the empty vector (mock) as a control or with human mutant SOD1G93A cDNAs and mouse NKA α1 siRNA or negative-control siRNA Western blotting was used to confirm that NKA α1 levels were knocked down (B) Cells were treated with vehicle or with human recombinant GPNMB (2.5 μg/ml) concomitantly with removing the serum ††p < 0.01 vs GPNMB treated group (Tukey’s test) (A) NSC34 cells were transfected with mouse NKA α1 siRNA or negative-control siRNA and then treated with the human recombinant GPNMB (2.5 μg/ml) (B) The protein levels of NKA α1 were quantified relative to β-actin (C) The protein levels of phosphorylated-Akt were quantified relative to total-Akt (D) The protein levels of phosphorylated-ERK1/2 were quantified relative to total-ERK1/2 (E) 661W cells were plated at 1.5 × 104cells/well in 24-well plates and incubated overnight Negative-control siRNA and NKA α1 siRNA transfected for 24 h and then cells were treated with PBS or human recombinant GPNMB (F) The protein levels of NKA α1 were quantified relative to β-actin (G) The protein levels of phosphorylated-Akt were quantified relative to total-Akt (H) The protein levels of phosphorylated-ERK1/2 were quantified relative to total-ERK1/2 The concentration used in this study was higher than the amount of GPNMB in the CSF or serum GPNMB is highly expressed in motor neurons and astrocytes in the spinal cord 2.5 μg/ml recombinant GPNMB may be sufficient for binding to alpha subunits of NKA Both NKA inhibitors and NKA α1 siRNA blocked the neuroprotective effects of GPNMB against stress-induced cell death and activation of the PI3K/Akt and MEK/ERK pathways via GPNMB binding NKA α1 siRNA transfection caused slight increases in the phosphorylation of Akt and ERK in the recombinant GPNMB-treated group but not to the extent that occurred when cells were transfected with negative-control siRNA These results suggested that the PI3K/Akt and MEK/ERK pathways may be slightly activated by recombinant GPNMB via the remaining NKAα1 after siRNA transfection because the siRNA was not 100% efficient the NKA α3 subunit may also be involved in the PI3K/Akt and MEK/ERK pathways because NKA α3 was not knocked down in this examination these results suggested that the extracellular fragment of GPNMB had neuroprotective effects and activated the PI3K/Akt and MEK/ERK pathways via NKA supporting the possibility that NKA may act a novel receptor of the GPNMB extracellular fragment these reports suggest that the activation of PI3K/Akt and MEK/ERK pathways by the extracellular fragment of GPNMB may be associated with FGFR signaling via NKA the RGD motif of the extracellular fragment of GPNMB may bind to the extracellular domain of NKA α1 which would then lead to further signaling through the PI3K/Akt and MEK/ERK pathways Further studies will be required to identify GPNMB extracellular fragment-specific binding sites and to investigate the interaction between GPNMB and NKA in vivo we have shown that the extracellular fragment of GPNMB associates directly with NKA alpha subunits we suggest that the NKA may function as a novel “receptor” of the GPNMB extracellular fragment and as an additional therapeutic target for various cancers and/or ALS This method allowed us to analyze all the membrane proteins in the cell and organelle membranes MPL was prepared by fusing the membrane fraction of a cellular lysate from mice whole brains of mice with liposomes The extracellular fragment of GPNMB was immobilized with N-hydroxysuccinimide-activated sepharose 4 fast flow (S4F) all the contents were placed in an empty column and the solution was stored as a flow-through fraction The solution was blocked with 0.1 M Tris-Cl (pH 8.5) and the GPNMB extracellular fragment immobilization carrier (GPNMB-S4F) was obtained A carrier subjected only to blocking was prepared as a control immobilization carrier (C-S4F) Unbound MPLs were washed away and the targeted receptors or protein dominant MPLs were eluted The elution was separated by electrophoresis Protein bands were detected through the differential analysis of SDS-PAGE profiles of the MPLs and were identified using MALDI-TOF-MS or LC-FT-MS a neuroblastoma and spinal cord hybrid cell line NSC-34 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Sigma-Aldrich USA) containing 10% fetal bovine serum (FBS; Valeant 100 U/ml penicillin and 100 μg/ml streptomycin (Meiji Co Japan) in a humidified atmosphere containing 5% CO2 at 37 °C The cells were passaged by trypsinization every 4 days and maintained in a 10 cm dish (BD Biosciences Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions NSC34 cells were plated at 1.4 × 105cells/well in 6-well plates (BD Biosciences) and incubated overnight The cell lysates were incubated with mouse anti-NKA α1 antibody or normal mouse IgG (Santa Cruz Biotechnology) A mixture of equal parts of a protein sample and sample buffer with 20% 2-mercaptoethanol (Wako The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF the primary antibody was goat anti-GPNMB antibody (1: 500 USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000 The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar® LD; Wako) The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film NSC34 cells were treated with the human recombinant GPNMB (extracellular fragment; R&D Systems Inc.) or normal human IgG (R&D Systems Inc.) the primary antibody was used normal human IgG (1: 500 The secondary antibody was HRP-conjugated goat anti-human antibody (1: 50000 NSC-34 cells were seeded at 3.5 × 104cells/well in a Lab-Tek II Chamber slide (Thermo Fisher Scientific) and incubated overnight cells were fixed in 4% paraformaldehyde (PFA) for 15 min cells were incubated overnight at 4 °C with primary antibody cells were incubated at room temperature with Alexa-488 or Alexa-568-labeled secondary antibody (1: 500 Confocal images were obtained using Fluoview (Olympus NSC-34 cells were transfected with 100 nM of mouse NKA α1 specific RNAi oligo or negative-control RNAi oligo (Nippon Gene Co. Japan) by using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for 48 h according to manufacturer’s recommendations Sequences included the NKA α1 small interfering RNA (siRNA) sense strand cells were treated with the human recombinant GPNMB or PBS for 1 h and fixed and blocked as the same as mentioned above The cells were incubated overnight at 4 °C with primary antibody anti-human IgG antibody (1:500 cells were incubated at room temperature with Alexa-488 or Alexa-546-labeled secondary antibody (1:500) for 1 h a total of at least 200 cells per condition were counted using image-processing software (Image-J ver NSC34 cells were seeded at approximately 3.5 × 104 cells per well in 24-well plates (BD Biosciences) and incubated for 24 h The cells were treated with PBS or the human recombinant GPNMB were used to investigate whether the extracellular fragment of GPNMB activates the PI3K/Akt and MEK/ERK pathways through NKA NSC34 cells were washed with PBS once in order to remove dead cells The NSC34 cells were then lysed using a RIPA buffer (50 mM Tris HCl and 1% Igepal CA-630) together with protease inhibitors and a phosphatase inhibitor cocktail (Sigma-Aldrich) The lysate was centrifuged and the supernatant was collected Protein concentration was determined from a standard curve of bovine serum albumin (BSA) with a BCA protein assay kit (Thermo Fisher Scientific) A mixture of equal parts of a protein sample and sample buffer with 20% 2-mercaptoethanol was subjected to SDS-PAGE The separated proteins were then transferred onto a PVDF the following primary antibodies were used: rabbit anti-phospho-Akt (Ser473 The secondary antibodies were HRP-conjugated goat anti-mouse and HRP-conjugated goat anti-rabbit antibody (1: 2000 The immunoreactive bands were visualized using ImmunoStar® LD The band intensity was measured using LAS-4000 Data are presented as the means ± standard error of the mean (S.E.M.) Statistical comparisons were made using a two-tailed paired student’s t-test or one-way ANOVA followed by Dunnett’s test and Tukey’s test with p < 0.05 indicating statistical significance Glycoprotein nonmetastatic melanoma protein B extracellular fragment shows neuroprotective effects and activates the PI3K/Akt and MEK/ERK pathways via the Na+/K+-ATPase Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice Hematopoietic growth factor inducible neurokinin-1 type: a transmembrane protein that is similar to neurokinin 1 interacts with substance P Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model Glycoprotein nonmetastatic melanoma protein B a potential molecular therapeutic target in patients with glioblastoma multiforme Molecular cloning of a dendritic cell-associated transmembrane protein that promotes RGD-dependent adhesion of endothelial cells through recognition of heparan sulfate proteoglycans Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion The PKD domain distinguishes the trafficking and amyloidogenic properties of the pigment cell protein PMEL and its homologue GPNMB is expressed in low-metastatic human melanoma cell lines and xenografts Clinical significance of glycoprotein nonmetastatic B and its association with HER2 in breast cancer Distinct high-profile methylated genes in colorectal cancer DC-HIL/glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells Microphthalmia transcription factor regulates the expression of the novel osteoclast factor GPNMB Expression of glycoprotein non-metastatic melanoma protein B in cutaneous malignant and benign lesions: a tissue microarray study The potential of GPNMB as novel neuroprotective factor in amyotrophic lateral sclerosis The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and gamma-secretase ADAM10 releases a soluble form of the GPNMB/Osteoactivin extracellular domain with angiogenic properties Osteoactivin fragments produced by ectodomain shedding induce MMP-3 expression via ERK pathway in mouse NIH-3T3 fibroblasts A new rapid and comprehensive peptidome analysis by one-step direct transfer technology for 1-D electrophoresis/MALDI mass spectrometry Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria Increase of the delayed rectifier K+ and Na(+)-K+ pump currents by hypotonic solutions in guinea pig cardiac myocytes A real-time screening assay for GIRK1/4 channel blockers The sodium pump alpha1 sub-unit: a disease progression-related target for metastatic melanoma treatment Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells Na,K-adenosine triphosphatase alpha1-subunit predicts survival of renal clear cell carcinoma The alpha1 subunit of the sodium pump could represent a novel target to combat non-small cell lung cancers Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer Increase in ouabain-sensitive K+-ATPase activity in hepatocellular carcinoma by overexpression of Na+ Global loss of Na,K-ATPase and its nitric oxide-mediated regulation in a transgenic mouse model of amyotrophic lateral sclerosis The Na/K-ATPase is obligatory for membrane anchorage of retinoschisin the protein involved in the pathogenesis of X-linked juvenile retinoschisis Identification of a pool of non-pumping Na/K-ATPase Effects of Na/K-ATPase and its ligands on bone marrow stromal cell differentiation Binding of Src to Na+/K+-ATPase forms a functional signaling complex Cell signaling associated with Na(+)/K(+)-ATPase: activation of phosphatidylinositide 3-kinase IA/Akt by ouabain is independent of Src Met5-enkephalin-induced cardioprotection occurs via transactivation of EGFR and activation of PI3K Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction GPNMB enhances bone regeneration by promoting angiogenesis and osteogenesis: potential role for tissue engineering bone The FGFR/MEK/ERK/brachyury pathway is critical for chordoma cell growth and survival Neural cell adhesion molecule modulates mesenchymal stromal cell migration via activation of MAPK/ERK signaling The ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase Compartmentalizing proximal FGFR1 signaling in ovine placental artery endothelial cell caveolae suppresses motor neuron damage in in vitro and in vivo amyotrophic lateral sclerosis models Download references This study was supported by a grant from the Ministry of Education Japan: a Grant-in-Aid for Scientific Research (C) (25460103) contributed to experiments on searching the receptor for the extracellular fragment of GPNMB participated in design of study and analysis data contributed to design of the study and wrote the paper with Y.O The authors declare no competing financial interests Download citation Sign up for the Nature Briefing newsletter — what matters in science Omu vana kutamununa ngendeseso zokugwana mahundiro govakandindate valitjangese nokupulisira nombunga politika dikaze nye monokampaign yiyo nye zina kutanta ezi ngendeseso zokutulisapo vakandindate melikwamo lyangesi.  Ngendeseso nazivatera mokuruganesa yimbapira yoyinzi morwa kuturamo mahundiro omu hena tuna hara kutunda monomukweyo dakarapo kovakandindate ndi kovahorowoli ose kuyagwanekera nyamwetu pouruwo yoyidigu – ngesi siruwo sokuruganesa unkurungu ntani magano gomawa aga tuna kara nago naga ninkisa asi tuze mongendeseso zetu zopaudemokoli.  Apa nye tuna kugava noforoma edi kupitira ponongodi nayininkisa asi vakandindate momaruha nagenye Eyi tayipenye mukumo udemokoli wetu omu tuna hara asi vantu wokulisiga siga valihameseremo.  ‘’ Eyi yokuruganesa konongodi hawe nayikara asi kapi tomana siruwo unene mokutumbura eyi ono hara Eyi kuna kuretesapo hena mutaro nye gomuwa unene ntani vantu nawo vahepa kuyihuguvara ntani ngendeseso zomahoroworo vahepa kuzimona asi zina magnurukire nkenye ogu mokuzitarurura,’’ yimo ana kutanta komisare gumwe nage gomahoroworo Gerson Tjihenuna.  Age kakahuyungire kahuyungire pedeuro lyowo vana kukaruganesa elikwamo eli ponongodi mokupitira moECN moutano kaupwireko Edeuro eli kalikere lyokudeura ntani kutwaramo ava vana kara asi tavakayiriugana rambangako novakarelipo wono,mbunga politika nawo vahepa kukara noudivi wangesi.  Age kahuguvaresere vantu asi hawe kuna kara ekeverero lyouhunga poyininbke yangesi ose kuvhura nye kukeverera nombudi detu dongendeseso zomahoroworo.  Age katanterere vakarelipo wonombunga politika asi eyi yina kara mulyo unene mongendeseso zomahoroworo getu moNamibia ‘’ Eyi kuna kara asi kapi yina kara tupu yokuhuyunga Eyi kuna kara yomutaro ntani kuna kuza komeho yina kara asi yomapuliro getu omu natugwanekera novantu vetu muhoko gwetu nayina nokomeho oko.  Ose kuna kuyimona asi nkenye mbunga politika ntani novakandindate vawo kuna kara nehuguvaro ntani nokuvapa siruwo sokulifana morwa mongendeseso zoudemokoli tuna kara ngesi.  Posiruwo sooso muhuyungilipo ECN Mulauli 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Volume 9 - 2022 | https://doi.org/10.3389/fmats.2022.877755 Surface modification of hemocompatible copolymers on silicone elastomers (SEs) is crucial for the long-term use of medical devices Both physical adsorption and chemical conjugation are important for modification of SE Oxygen plasma treatment is widely used to produce silanol groups on SE for silane coupling the plasma reaction is difficult to apply to the surface modification of three-dimensional complex devices This study demonstrated an appropriate and efficient method with alkaline solution for producing silanol groups on SE for modifying phosphorylcholine-based copolymer with organosilane (cross-MPC copolymer) A 2.5 wt% aqueous solution of potassium hydroxide (KOH) was effective in producing silanol groups and for coating the cross-MPC copolymer we successfully modified the cross-MPC copolymer on the inner surface of SE tubes after pretreatment with the 2.5 wt% KOH aqueous solution and the copolymer film was coated homogeneously The cross-MPC copolymer film on SE was stable for one month under fluidic condition with a shear stress of 3.2 Pa The hollow fiber membrane with the polymer coating inhibited blood coagulation after one week implantation with extracorporeal circulation device using a goat pretreatment of SE using an alkaline solution is an appropriate method for producing silanol groups for coating the cross-MPC copolymer by silane-coupling reaction Currently, the usage time of ALs with membrane-type oxygenators is limited to several hours in clinical use due to the poor durability; however, long-term durability is required to apply as a bridge for the transplantation of lungs (Duy Nguyen et al., 2021). Furthermore, there has been an increasing demand for the continuous use of oxygenators because of the Covid-19 pandemic (Sanford et al., 2020; Raasveld et al., 2021) The coated cross-MPC copolymer on SE inhibited the adsorption of plasma proteins for three months we have applied the liquid-phase process with an alkaline solution for the surface functionalization of silicone elastomer and evaluated the functions such as hemocompatibility and stability of coated film of the phosphorylcholine-based copolymer containing organosilane This liquid-phase process has various advantages such as mass production and low cost compared with vacuum-phase processes the durability of the cross-MPC copolymer film on SE was evaluated in vitro under dynamic flow condition for one month The hemocompatibility of the cross-MPC copolymer film-coated hollow fiber membrane as a model of medical devices was tested using a goat blood circuit for a one-week in vivo experiment The cross-MPC copolymer was provided by NOF Corporation, Japan, and it was synthesized via free radical copolymerization of MPC, MPTSSi, and MPTMSi. The composition ratio of MPC, MPTSSi, and MPTMSi in the cross-MPC copolymer was 54, 27, and 19 mol%, respectively (Nagahashi et al., 2015) This ratio in the cross-MPC copolymer had been optimized to coat polymer film well with good inhibition for protein adsorption Sheets (1.0 × 1.0 × 0.1 cm) and tubes (outer diameter = 0.9 cm; inner diameter = 0.6 cm length = 10.0 cm) made of SEs were fabricated by Fuji Systems Co The membrane (1.0 × 1.0 cm) was fabricated from hollow fibers with an outer diameter of 0.4 mm by holding hollow fibers at a constant spacing of 0.4 mm using a string and sodium dodecyl sulfate (SDS) were purchased from FUJIFILM Wako Pure Chemical Corporation Ltd and phosphate-buffered saline (PBS) were purchased from Kanto Chemical Co. fluorescein isothiocyanate-labeled BSA (FITC-BSA) and glutaraldehyde (GA) were purchased from Sigma–Aldrich (St The µ-BCA protein assay kit was purchased from Thermo Fisher Scientific Inc The blood collection tube with 0.2 ml 3.2% citric acid for preparing platelet-rich plasma was purchased from Nipro Corporation (Osaka The coating process of the crosslinking-type MPC-copolymer via pretreatment of its chemical structure are shown in Figure 1. The SE substrates were ultrasonically washed in ethanol for 15 min, followed by drying through air blowing. Subsequently, the substrates were immersed in an aqueous solution of NaOH or KOH for 1 h with gently shaking on shaker under different conditions, such as the type, concentration, and temperature (Table 1) substrates were treated under the condition of 20 W at 600 mTorr for 2 min (PDC-001 the substrates were washed sufficiently with pure water Schematic coating procedure for the cross-MPC copolymer film on silicone elastomer (SE) surface (A); Chemical structure of the cross-MPC-copolymer (B) Alkaline pretreatment conditions on silicone elastomers for cross-MPC copolymer coating A methanol solution containing 0.1 wt% polymer was mixed with an aqueous solution of 0.1 M acetic acid (acetic acid/polymer solution: 10/90 The sheets and hollow fibers were coated with the cross-MPC copolymer film by dipping in the coating solution for 2 h To coat the inner surface of the tube with the cross-MPC copolymer the tubes were filled with the polymer and were sealed with forceps and tubes were dried in a vacuum chamber for 1 h and then heated at 70°C for 3 h to dehydrate To evaluate the uniformity and thickness of the cross-MPC copolymer film via fluorescence microscopy, R6G was stained on the polymer film (Wang et al., 2005) the cross-MPC copolymer-coated SE sheets were immersed in water for 1 h and then they were soaked in an aqueous solution of 0.01 wt% R6G at room temperature for 30 s they were immersed in water for 30 min to wash the extra R6G The sheets were observed via fluorescence microscopy (Axioscope 2 Plus The fluorescence images were captured using a charge-coupled device (CCD) camera (VB-7010 and fluorescence intensity was analyzed using an imaging software (ImageJ The surface morphology of the cross-MPC copolymer-coated sheets was observed via scanning electron microscopy (SEM) at an acceleration voltage of 5 kV (JSM-7000Fm The cross-MPC copolymer film was coated with osmium (Neoc-ST The elemental composition for evaluating the film stability and silanol groups was characterized via XPS (JPS-9010MC Elemental analysis of poly-SE surfaces was conducted via XPS equipped with a 10 kV magnesium Kα radiation source at an electron take-off angle of 90° from the surface where FIpoly-SE is the fluorescence intensity of the cross-MPC copolymer-coated SE and FISE is the fluorescence intensity of SE without polymer coating the SE sheets (1.0 × 4.0 × 0.1 cm) were incubated in 0.3 mg/ml HSA and 0.3 mg/ml HPF at 37°C for 1 h The HSA and HPF adsorbed sheets were washed with PBS under magnetic stirring to remove the unabsorbed protein Then the 3 pieces of protein adsorbed sheets were immersed in 6 ml of PBS containing 1 wt% SDS ultrasonic waves were applied at room temperature for 5 min to detach the adsorbed HSA and HPF The concentration of HSA and HPF in the SDS solution was determined using the µ-BCA protein assay the 3D model was segmented into tetrahedrons The grid/mesh was fabricated using 1.3 × 107 elements (surface of tetrahedral structure) and 2.3 × 106 nodes (grid points of the element corner) As the parameters of the circulating fluid the density and dynamic viscosity of PBS were set to 1723 kg/m3 and 0.8882 mPa s The pressure of the outlet was 0 mmHg and the flow rate of the inlet was set to 8 L/min The steady state flow was assumed with no-slip condition of the surface and non-gravity effects and it was set as laminar flow because the Reynolds number was 3659 lower than 4000 Schematic circulating system for evaluating the stability of the polymer film: (A) The tubing with three pieces of silicone elastomer (SE) sheets of 1.27 × 1.00 cm located inside at a distance of 1.00 cm (B) Illustration of the liquid flow circulation circuit for evaluating the stability of the cross-MPC copolymer film on different pretreated SE sheets a) oxygen plasma treated-silicone elastomer (OPT-SE) b) 2.5 wt% KOH treated silicone elastomer (2.5K-SE) and c) without pretreated silicone elastomer (SE) The liquid circulation circuit was filled with PBS and placed in an incubator at 37°C The flow rate was controlled at 8 L/min (2500 rpm) using a centrifugal pump then the SE sheets were rinsed with water and vacuum dried in a desiccator overnight The atomic ratio of N/C (N in the MPC unit/C in the polymer film and SE) of the surface of the sheets was evaluated via XPS APTES was used for the reaction of the silanol group and the reaction between the cross-MPC-copolymer and the SE pretreated with alkaline was investigated the OPT-SE sheets were modified with APTES as a control The pretreated SE sheets were dipped into a 0.5% APTES solution (in ethanol) for 1 h at room temperature the APTES-modified sheets were rinsed with ethanol and dried in vacuum overnight in a desiccator and the atomic ratio of N/Si was calculated from the elemental composition of the SE after APTES modification Platelet-rich plasma (PRP) was freshly prepared from the whole blood of a goat (Japan Saanen goat The whole blood (1.8 ml) was collected using a syringe and injected into a blood collection tube it was centrifuged at 184.5 × g (1000 rpm) for 30 min in a refrigerated compact centrifuge (himac-CF7D2 The mixture of plasma supernatant containing platelets and a small amount of red blood cells was transferred into another sterile tube and centrifuged at 1660.2 × g (3000 rpm) for 5 min at 4°C The PRP concentration was 1.1×108 ± 1.2×107 cell/mL and the cross-MPC copolymer-coated SE (poly-2.5K-SE) sheets were incubated in PRP at 37°C for 1 h and SE sheets were rinsed with PBS to remove unadhered platelets The PRP incubation for stability evaluation was same as polymer coated SE The adhered platelets were fixed with 2.5 wt% GA in PBS for 30 min at room temperature and 100% ethanol for 30 min in order the sheets were dried in a vacuum overnight and stored in a desiccator The platelets that adhered on the SEs were observed via SEM at an acceleration voltage of 5 kV (JSM-7000Fm Japan) after coating with osmium using a plasma coater (A) Development of a chamber of acrylic plates pasted with membranes made of hollow fibers (HFM) of 1.0 × 1.0 cm Eight pieces of HFM were coated with the cross-MPC copolymer (poly-HFM) (B) Schematic illustration of in-vivo experiment for evaluating the blood compatibility The chamber was connected to the ECMO system in series The centrifugal pump in ECMO system was controlled from 1.0 to 2.0 L/min The pulmonary artery was connected to sutured cannulas of inflow and outflow to the left atrial appendage An extracorporeal membrane oxygenation (ECMO) system (EMERSAVE Terumo Corporation) was attached to the sutured cannula for extracorporeal circulation The chamber was attached by cutting the tube in the circulation system between the centrifugal pump and AL The blood flow was measured using a flow meter and controlled using a pump controller between 1.0 and 2.0 L/min The activated clotting time (ACT) in the blood was adjusted to less than 250 using heparin the chamber was removed from the ECMO system the poly-HFMs and HFMs were fixed with 2.5% GA and observed via SEM but the etching by O2 plasma has occurred more severe than 2.5K We confirmed that the etching thickness of O2 plasma was around three times higher than that of KOH solution although the etching occurred in less than several nanometers In summary of the alkaline solution pretreatment the increase in surface roughness was caused by an increase in the concentration of the alkaline solution and the reaction temperature the 2.5K alkaline treatment was the most appropriate condition for alkaline treatment and was applied for various experiments FIGURE 4. Characterizations of the polymer film coated under different conditions (Table 1) Fluorescence intensity of the adsorbed R6G on the polymer film (A); Relative fluorescence intensity of adsorbed FITC-BSA on the polymer-coated surface (B) Results are presented as mean ± SD (n = 9) (C) Optical microscope images for surface morphology of SEs (i) without treatment and *** indicate p < 0.05 Here, the applicability of alkaline solution pretreatment on the inner surface of tubular-shaped materials was evaluated. The inner surfaces of the SE tubes (inner diameter: 0.6 cm) were pretreated with 2.5 wt% alkaline solution. Thereafter, the pretreated tubes were coated with the polymer. The polymer-coated tubes were cut into small pieces to evaluate the polymer film coating and protein adsorption (Figure 5A). As shown in Figure 5B the fluorescence intensities of adsorbed R6G at the different parts of the inner tubes were not significantly different indicating that the polymer-coated film on the alkaline-treated tube was homogeneous Characterization of the polymer coated in silicone elastomer tube (inner diameter: 0.6 cm) (A) Procedure for the characterization of the polymer-coated tubes with an alkaline solution (2.5K) The tubes were cut into small pieces before staining them with Rhodamine 6G (R6G) and FITC-BSA/BSA incubation Fluorescence intensity of the adsorbed R6G (B) and relative fluorescence intensity of the adsorbed FITC-BSA (C) on the polymer-coated tube with alkaline pretreatment (2.5 K) Results are presented as mean ± SD (n = 3) Additionally, protein adsorption was evaluated by measuring the RFI with FITC-BSA/BSA incubation. As shown in Figure 5C after polymer coating on the alkaline pretreated tube the entire area in the SE tube prevented protein adsorption with a homogeneous polymer film These results indicate that pretreatment with an alkaline solution is appropriate for the coating of cross-linked copolymers on tubular-shaped materials or medical devices with complex structures the average value of WSS was 3.2 Pa Stability evaluation of the coated polymer film on the silicone elastomer (SE) sheet in a fluidic circulating system (A) Color maps for cross section view of side (i) and top view (ii) of wall shear stress (WSS) by computational fluid dynamics (CFD) model in the tubing (inner diameter = 1.27 cm; outer diameter = 1.59 cm) The inlet flow rate was set as 8 L/min without outlet pressure (B) The N/C (nitrogen comes from the polymer/carbon) ratio of the coated-polymer film on oxygen plasma treated (poly-OPT) 2.5 K alkaline treated (poly-2.5K) The data was measured every week for one month Results are presented as mean ± SD (n = 6) and *** denote p < 0.05 (C) Evaluation of the silanol group on the surface of SE The silanol group was reacted with APTES after treatment with 2.5 K (APTES-2.5K) and oxygen plasma (APTES-OPT) and then N/Si (nitrogen comes from APTES/silicon) was measured via XPS Thus, the results of the CFD analysis indicated that the evaluation setup composed of the fluidic circulation system shown in Figure 2B was reliable for evaluating the stability of the coated polymer film After aging under fluidic condition, the atomic composition of N in the MPC component, C in the SE, and cross-MPC copolymers were analyzed via XPS. Figure 6B shows the relationship between the atomic ratio of N/C and the aging time The remaining polymer films on OPT SE (poly-OPT) and 2.5 wt% KOH solution-treated SE (poly-2.5K) at each aging time exhibited a similar decreasing trend the remaining polymer film on the non-treated FSE (poly-SE) significantly decreased after aging for 1 month and had significant difference from the poly-2.5K Although the continuous flow was circulated for 4 weeks the polymer film coated after pretreatment with the alkaline solution and oxygen plasma remained on the SE the N/C ratio for indicating the remaining polymer of poly-OPT and poly-2.5K was slightly decreased owing to the degradation of the polymer The treatment using KOH solution and OPT produced silanol groups Considering the relationship between the stability of the polymer film and the density of the silanol group the stability was successfully enhanced by increasing the density of the silanol group the 2.5K alkaline treatment obtained sufficient silanol groups on the SE surface to provide conjugatable sites for coating the cross-MPC copolymer These results indicate that the poly-2.5K-SE inhibited protein adsorption and platelet adhesion (A) Relative HSA and HPF adsorption on the uncoated SE as well as the poly-2.5K-SE via µ-BCA assay (B) Hemocompatibility evaluation under static condition SEM micrographs of SE and poly-2.5K-SE after incubation in PRP for 1 h at 37°C (C) Hemocompatibility evaluation after aging under fluidic circulation for 4 weeks and (iv) poly-SE after incubation in PRP for 1 h at 37°C To confirm the hemocompatibility of the coating film of cross-MPC copolymer after aging under fluidic circulating for 4 weeks which are the same condition shown in Figure 6, the SE, poly-OPT-SE, poly-2.5K-SE and poly-SE substrates were incubated in PRP for 1 h at 37°C, and then observed by SEM. The PRP incubated SE was conducted as control. The SEM images of adhered platelet are shown in Figure 7C The results indicated that the poly-2.5K and poly-OPT still had good inhibition of platelet adhesion after aging by fluidic circulating with PBS for 4 weeks the poly-SE had many adhered platelets on the surface These results were suggested that high-density of silanol group produced by KOH solution on silicone elastomer improved the stability of polymer film and keep hemocompatibility for long time this surface modification can be applied to medical devices for long-term use Thicker thrombi are induced by the activation of platelets leading to the formation of the thrombus on the HFM (A) Photograph of the chamber with 16 pieces of membranes made of the hollow fiber (HFMs) after one week of implantation in goat The upper part of the membranes was coated with the polymer film (poly-HFM) (B) The SEM images of the hollow fiber coated with the polymer film (i ii) and those without polymer coating (iii This result was consistent with the results of protein adsorption and platelet adhesion in the in-vitro evaluation, as shown in Figure 7. The activated platelets on SE contributed to blood coagulation and led to burnt red thrombus, as shown in Figure 8A. In addition, the thrombus filled the space between the hollow fibers (Figure 8Biii) because they formed easily under a slow flow rate These results implied that the blood did not coagulate on the poly-HFM The poly-HFM treated with KOH solution exhibited good hemocompatibility for in-vivo implantation for one week under fluidic conditions the cross-MPC copolymer was coated via a silane coupling reaction on functionalized SE The type and concentration of the alkaline solution as well as the reaction temperature were optimized to achieve good polymer film formation and effective inhibition of protein adsorption on the coated polymer films The increased protein adsorption on the polymer-coated SE was induced by the stronger etching resulting from high concentration and high temperature of KOH and NaOH solutions The 2.5 wt% KOH solution at room temperature performed better compared to other pretreatment conditions as evidenced by less protein adsorption on polymer-coated surface and good polymer film formation The optimum conditions for alkaline treatment were applied to the coating on the inner surface of the SE tube There was no difference in the fluorescence intensity in different areas which indicated excellent homogeneity of the coated polymer film on the SE surface in tube Although the polymer films were removed gradually under the shear stress (3.2 Pa) of the fluid condition the stability of the cross-MPC copolymer film on the SE pretreatment with 2.5K alkaline solution for one month was comparable to that of the polymer film on SE pretreated with OPT It was established that the increase in the stability of the cross-MPC copolymer film on SE depended on the amount of the silanol groups The results of platelet adhesion on 4-weeks aging under fluidic condition shows that the stability of cross-MPC copolymer film on the SE pretreated with 2.5K alkaline solution was good enough to inhibit platelet adhesion For in vivo experiments using a goat with ECMO the cross-MPC copolymer-coated HFM effectively inhibited blood coagulation for one week under blood flow coating film of the cross-MPC copolymer via silane coupling reaction on functionalized silicone elastomer by KOH solution could keep good hemocompatibility for long time This is beneficial for developing improved interfaces for medical devices The raw data supporting the conclusion of this article will be made available by the authors The animal study was reviewed and approved by the University of Tokyo Animal Experiment Committee Written informed consent was obtained from the owners for the participation of their animals in this study writing—original draft; SH: investigation writing—review & editing; KU: investigation writing-review & editing; RY: methodology for chamber with hollow fiber membranes resources for silicone elastomer substrates; TO: investigation for in vivo experiment; MA: supervision investigation for surgery of in vivo experiment writing—review & editing; TI: supervision writing—review & editing; MT: conceptualization This work was supported by Japan Agency for Medical Research and Development (AMED) (grant number 18hm0102048h0002) (MT and Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science (grant number 20K17577) (SH) The funds received for open access publication fees from our other grants RY was employed by the company Fuji Systems Corporation 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 A part of this work was conducted at Advanced Characterization Nanotechnology Platform of the University of Tokyo, supported by “Nanotechnology Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We would like to thank Editage (www.editage.com) for English language editing The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmats.2022.877755/full#supplementary-material Activation of PVDF Membranes through Facile Hydroxylation of the Polymeric Dope CrossRef Full Text | Google Scholar Behavior of Plasma-Treated Elastomeric Polydimethylsiloxane Coatings in Aqueous Environment CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar Surface Etching of Silicone Elastomers by Depolymerization CrossRef Full Text | Google Scholar Performance of Carbohydrate-Modified Fused-Silica Capillaries for the Separation of Proteins by Zone Electrophoresis CrossRef Full Text | Google Scholar Phosphorylcholine Coating of Extracorporeal Circuits Provides Natural protection against Blood Activation by the Material Surface PubMed Abstract | CrossRef Full Text | Google Scholar The Roles of Membrane Technology 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: Madoka Takai, dGFrYWlAYmlzLnQudS10b2t5by5hYy5qcA== Metrics details Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells to identify the keratinized mucosa-specific basement membrane components immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization These findings indicate that α112:α556 (IV) network which is the only network that includes the α6(IV) chain is one regulator of KRT10 expression in keratinization of oral mucosal epithelium the understanding of the mechanisms behind the process of gingiva keratinization is essential for the development of novel techniques and materials for keratinized gingival tissue reconstruction and regeneration the mechanisms regulating keratinization of gingiva still remain unclear It is well known that mutations in type IV collagen α3 to α5 chains causes Alport’s syndrome associated with glomerulonephritis sensorineural deafness and eye abnormalities we hypothesized that the difference in keratinization of palatal and buccal mucosa could be associated with the composition of the basement membrane We herein focused especially on type IV collagen and found that α5(IV) and α6(IV) chains were highly expressed in keratinized oral mucosa Loss-of-function analysis using Col4a6 knock out mice (Col4a6-KO mice) and gene expression knockdown with siRNA indicated that α112:α556 (IV) network plays an important role in the keratinization of oral mucosa epithelial cells Comparison of palatal and buccal mucosa Histological analysis of oral mucosa in 8-weeks-old mice HE staining (A) and IHC staining for KRT10 (green) (B) or KRT14 (C) was performed using coronal sections of mouse head Nuclei were counterstained with DAPI (blue) Boxes indicate the area shown at higher magnification in the right panels Note that KRT10 is highly expressed in keratinized mucosa whereas KRT14 is expressed in basal cells of both keratinized and non-keratinized mucosa (D) mRNA expression levels of Krt1 and Krt10 in palatal and buccal mocosa was measured by real time RT-PCR The expression of each gene was normalized to that of S29 ribosomal RNA Bars represent the mean values and standard deviation (+/−SD) (n = 3) Results are representative data of at least three independent experiments (E) TEM image of palatal mucosa (left) and buccal mucosa (right) Boxes indicate the area shown at higher magnification Analysis of the six alpha chains of type IV collagen in the basement membrane of keratinized and non-keratinized mucosa α6(IV) (K,L) was performed to compare the expression of these proteins in keratinized (A,C,E,G,I,K) and non-keratinized mucosa (B,D,F,H,J,L) Yellow arrows indicate positive signals for each antibody in the basement membrane Note that α5(IV) and α6(IV) chains are highly expressed in keratinized mucosa Results are representative of at least three independent experiments Expression analysis of α6(IV) and KRT10 during mouse embryonic development The mucosal samples of mice embryos at E12.5 (A,E,I,M) E16.5 (C,G,K,O) and E18.5 (D,H,L,P) were collected HE staining of the keratinized (palatal) mucosa is shown in A–D Boxes indicate the area shown at higher magnification in the low panels (E–L) show single staining for α6(IV) (red) and KRT10 (green) in palatal mucosa Comparison of keratinized mucosa between new-born and aged WT and Col4a6-KO mice (A,C) IHC staining for KRT10 (green) in palatal mucosa of WT and Col4a6-KO mice in new-born (A) and 28-week-old aged mice (D) The percentage of positive fluorescent signal for KRT10 in the area of palatal mucosa of new-born and aged mice is shown in graphs B and D (E–F) IHC staining for KRT14 (green) in palatal mucosa of WT and Col4a6-KO mice in new-born (E) and 28-week-old aged mice (F) Note that KRT14 levels are higher in newborn WT mice compared to Col4a6-KO mice the levels of KRT14 are identical in both WT and Col4a6-KO mice we concluded that α556(IV) protomer is absent and that α112:α556 network is not synthesized in keratinized mucosa of Col4a6-KO mice Expression analysis of COL4A6 and keratin markers during epithelial keratinization in vitro hGECs were seeded in the ThinCert cell culture inserts (3D culture) KRT10 (C) and INV (D) were measured by real time RT-PCR These results indicate a novel role of α6(IV) as one of the regulators of keratinization of oral mucosal epithelium type IV collagen α1(IV) and α2(IV) chains were expressed equally in keratinized and non-keratinized mucosa and α3(IV) and α4(IV) chains were not detected in either of the tissues the results highlight the importance of type IV collagen α6 chain ablation of α6(IV) does not disrupt basement membrane assembly no notable phenotype in the kidney was observed this study is the first to demonstrate a phenotype of Col4a6 deletion in the keratinization of oral mucosa in mice we could not show the localization of Col4a6 mRNA by using in situ hybridization we could observe the increased Col4a6 mRNA level during epithelial keratinization of hGECs in the 3D culture model that epithelial cells can synthesize COL4A6 in oral mucosa we assume that epithelial cells are able to secrete α6(IV) chain which in turn could activate intracellular pathways that direct cell differentiation in an autocrine manner the epithelial-mesenchymal interaction could play fundamental roles in determining the differential expression of α6(IV) chain in keratinized mucosa we had performed a preliminary study using hGECs which were co-cultured with mesenchymal cells isolated either from keratinized or non-keratinized mucosa We observed that keratinization occurred only in hGECs co-cultured with mesenchymal cells from keratinized mucosa there may exist numerous factors activated by the epithelial-mesenchymal cell interaction that could induce the initial synthesis and secretion of α6(IV) chain in cells forming the keratinized mucosa It has been reported that the mutation of α5 chain in X-linked Alport’s syndrome caused the loss of α6 chain and failure of assembly of α556 protomer and α112:α556 network (Zheng there could also have an absence of α112:α556 network due to loss of α6 chain in the keratinized gingiva of Col4a6-KO mice Based on the fact that the development of epithelium is delayed in Col4a6-KO new-born mice and that cell proliferation marker (cyclin D1) was also decreased in Col4a6 silenced hGECs (data not shown) may provide a niche environment for keratinocyte stem cells and regulate keratinocyte proliferation and differentiation in oral mucosal epithelium the biological mechanism of α6(IV) chain on stem cells has not been fully understood such as immunoassay and cell receptor assay using the recombinant protein α556(IV) IHC analysis showed no difference in perlecan levels in keratinized gingiva and non-keratinized gingiva between new-born Col4a6-KO and WT mice Due to the large number of molecules in the basement membrane further investigations are necessary to allow a deeper insight on the role of other components of the basement membrane in the keratinization of oral mucosal epithelium our study demonstrated that α5(IV) and α6(IV) chains were highly expressed in keratinized mucosa Keratinization of oral mucosal epithelium of Col4a6-KO mice was decreased in new-born mice and aged mice down-regulation of COL4A6 in hGECs caused suppression of epithelial keratinization these data indicate that type IV collagen α6 chain is involved in KRT10 expression in keratinization of oral mucosal epithelium Col4a6-KO mice were then backcrossed with C57BL/6 J (Charles River) for ten generations The animal experiment protocols used in this study (OKU-2016495 OKU-2017051) were approved by Okayama University Animal Research Committee All animals were handled according to the guidelines of Okayama University Animal Research Committee the specimens were fixed with acetone for 20 minutes followed by incubation with primary antibodies at 4 °C overnight after blocking with 5% normal goat serum (Life technologies) containing 1% BSA (Sigma Monoclonal primary antibodies specific for type IV collagen (α1 [H11] All antibodies for type IV collagen were diluted to 1:100 except for H22 and b14-stained sections were treated with 6 M urea in 0.1 M glycine/HCl (pH 3.5) for 10 minutes and B66 for 1 min before blocking KRT1 (ab185629) and KRT14 (ab181595) were purchased from Abcam (Cambridge the specimens were incubated with secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG (Life technologies Alexa Fluor 488 donkey anti-rat IgG (Life technologies) or Alexa Fluor 647 anti-rabbit for 60 minutes at room temperature All images were taken by fluorescence microscope Quantification of KRT10 positive area in epithelium was based on fluorescent IHC images taken with BZ-700 microscope and analyzed with a BZ analyzer (Keyence The collected palatal and buccal mucosa were homogenized using PowerMasher II (Nippi total RNA was extracted using TRIzol® Reagent (Life technologies) according to a conventional method and purified using a PureLink® RNA Mini Kit (Life technologies) The total RNA from cultured cells was extracted and purified using PureLink® RNA Mini Kit The palatal and buccal mucosa were harvested from eight-week-old female c57BL/6 J mice The collected samples were fixed with 2% glutaraldehyde and 2% PFA overnight After post-fixation with 1% osmium tetroxide and dehydration with ethanol the samples were embedded in spurr low-viscosity embedding media (Polysciences Ultrathin sections were then cut using a diamond knife and microtome (LEICA EM UC7 The sections were double-stained with uranyl acetate and lead citrate and observed using a transmission electron microscope (TEM: H-7650 Human gingival epithelial cells (hGECs) were purchased from CELLnTEC advanced cell systems AG (Stauffacherstrasse hGECs were cultured in CnT-prime epithelial culture medium (CELLnTEC advanced cell systems AG the cells were seeded onto cell culture inserts in a 12-well plate (ThinCertTM Japan) using CnT-Prime 3D barrier medium (3D-medium the cells were seeded at 5.0 × 105 cells in the upper chamber with 0.5 mL of 3D-medium Additional 5 mL of 3D-medium was added to the lower chamber For the down-regulation of the Col4a6 gene 5 nM of siRNA targeting the Col4a6 gene (StelthTM SiCol4a6; Life Technologies) was transfected into hGECs using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions StelthTM RNAi Negative Control High GC Duplex (Life Technologies) was used as the negative control Transfected hGECs were seeded at 2.0 × 106 cells in the upper chamber with 0.5 mL of 3D-medium and 5 mL of 3D-medium was added to the lower chamber and 4 mL of 3D-medium was added only to the lower chamber and the cells were cultured for additional 7 days hGECs were lysed using M-PER (Mammalian Protein Extraction Reagent; Thermo USA) supplemented with a Protease Inhibitor Cocktail (Roche Cell debris were removed from lysates by centrifugation at 12,000 rpm for 10 min at 4 °C Protein concentration in the cell lysate was determined by using Pierce BCA Protein assay kit (Thermo) Twenty five micrograms of the total protein was separated in precast polyacrylamide gels (NuPage Life Technologies) by electrophoresis and then transferred onto polyvinylidene fluoride membranes (PVDF; GE Healthcare Life sciences Blots were blocked and incubated with primary antibodies against KRT 10 (ab76318 and incubated with goat anti-rabbit lgG-HRP (1:2000; Santa Cruz Biotechnology USA) or goat anti-mouse lgG-HRP (1:2000; Santa Cruz Biotechnology) for 1 h at room temperature The blots were developed using Forte western HRP Substrate (Millipore) and visualized with Image Quant LAS 4000 mini (Fujifilm The results obtained from quantitative experiments were reported as the mean values ± SD Statistical analyses were performed with one-way factorial ANOVA followed by Tukey’s multiple comparison tests or Student’s unpaired t-tests Oral keratinocyte stem/progenitor cells: specific markers molecular signaling pathways and potential uses Epithelial barrier and oral bacterial infection Derivation of keratinocytes from chicken embryonic stem cells: establishment and characterization of differentiated proliferative cell populations The relationship between the width of keratinized gingiva and gingival health Gingival condition in areas of minimal and appreciable width of keratinized gingiva Significance of keratinized mucosa in maintenance of dental implants with different surfaces Five-year evaluation of the influence of keratinized mucosa on peri-implant soft-tissue health and stability around implants supporting full-arch mandibular fixed prostheses Basement membranes: cell scaffoldings and signaling platforms The extracellular matrix: not just pretty fibrils Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development Basement membranes: Putting up the barriers The NC1 domain of collagen IV encodes a novel network composed of the alpha 1 and alpha 6 chains in smooth muscle basement membranes The distribution of type IV collagen alpha chains in the mouse ovary and its correlation with follicular development Organization and expression of basement membrane collagen IV genes and their roles in human disorders Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development Aging decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-beta1 Loss of expression of type IV collagen alpha5 and alpha6 chains in colorectal cancer associated with the hypermethylation of their promoter region Col4a1 mutations cause progressive retinal neovascular defects and retinopathy The nature and biology of basement membranes Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): implications for Alport syndrome Minor Type IV Collagen alpha5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1 Stem cell patterning and fate in human epidermis A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes Characterization of a unique technique for culturing primary adult human epithelial progenitor/“stem cells” Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease The importance of laminin 5 in the dermal-epidermal basement membrane Perlecan expression influences the keratin 15-positive cell population fate in the epidermis of aging skin Distinct target-derived signals organize formation Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot’s film method (2012) Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine COL4A6 is dispensable for autosomal recessive Alport syndrome Diversity of human plasma protein C inhibitor Download references Yasuko Tomono (Shigei Medical Research Institute Japan) for kind gift of antibody against α(IV) chains Masumi Furutani (Central Research Laboratory of Okayama University Medical School This work was supported by JSPS KAKENHI Grant Numbers JP15H0502618 and J16H0552418 and the Translational Research program; Strategic PRomotion for practical application of INnovative medical Technology (TR-SPRINT) from Japan Agency for Medical Research and Development Department of Oral Rehabilitation and Regenerative Medicine Okayama University Graduate School of Medicine Department of Molecular Biology and Biochemistry 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/s41598-018-21000-0 Metrics details Whole-organ regeneration has great potential for the replacement of dysfunctional organs through the reconstruction of a fully functional bioengineered organ using three-dimensional cell manipulation in vitro many basic studies of whole-tooth replacement using three-dimensional cell manipulation have been conducted in a mouse model Further evidence of the practical application to human medicine is required to demonstrate tooth restoration by reconstructing bioengineered tooth germ using a postnatal large-animal model we demonstrate functional tooth restoration through the autologous transplantation of bioengineered tooth germ in a postnatal canine model which was reconstructed using permanent tooth germ cells erupted into the jawbone after autologous transplantation and achieved physiological function equivalent to that of a natural tooth This study represents a substantial advancement in whole-organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine we demonstrated functional tooth restoration after transplanting bioengineered tooth germ in a postnatal large-animal model which was reconstructed using canine permanent tooth germ developed with the correct tooth structure after autologous transplantation into the jawbone the erupted bioengineered teeth showed satisfactory physiological function with respect to the biological response to mechanical stress and this response was equivalent to that of natural teeth This study highlights the feasibility of fully functional tooth restoration by autologous transplantation of bioengineered tooth germ (A) Schematic representation of the generation of reconstituted tooth germ (Illustration by R.N.) (B) Photograph (left) and histological image obtained by HE staining (centre) of permanent first molar tooth germ (M1) and phase contrast image of reconstituted tooth germ on organ culture day 2 (right) (C) Micro-CT images of natural tooth germ at 8 and 12 weeks after subrenal capsule transplantation Three-dimensional (3D) images are shown in the upper column and cross-sectional views are shown in the lower column (left) Histological analysis of the bioengineered tooth 12 weeks after subrenal capsule transplantation (right) Boxes indicate the area shown at higher magnification in the lower panels (D) Bioengineered tooth germ was reconstituted using single cells derived from molar tooth germ Micro-CT images (left) and histological image (right) of the bioengineered tooth 8 weeks after subrenal capsule transplantation and cross-sectional views are show in the lower column we adopted the autologous transplantation model in dogs at postnatal day 30 to reconstruct bioengineered tooth germ using the permanent premolar germs that were subsequently transplanted into the autologous lower jaw (A) Schematic representation of autologous transplantation methods for the bioengineered tooth using postnatal canine-derived tissue (Illustration by R.N.) (B) Photograph of the extracted deciduous molar (dM) with permanent premolar tooth germ (left) and isolation of the permanent premolar tooth germ (right) (C) Photograph (left) and histological image by HE staining (right) of the isolated permanent premolar tooth germ (D) Phase contrast image of the reconstituted canine tooth germ using epithelial tissue and mesenchymal cells derived from permanent premolar tooth germ after 2 days in organ culture (E) Photograph of the autologous transplantation of bioengineered tooth germ into canine lower jawbone (F) Oral photographs and CT images of the erupted bioengineered tooth at 180 days after transplantation: no transplantation group (left) natural tooth germ transplantation group (centre) and bioengineered tooth germ transplantation group (right) (G) Micro-CT images and histological analysis of the natural tooth group (upper) the natural tooth germ transplantation group (middle) and the bioengineered tooth germ transplantation group (lower) Histological analysis of the bioengineered tooth over the crown area and the periodontal tissue area SEM image of enamel (A) and dentin (B) of the natural tooth the erupted tooth formed by the transplantation of natural tooth germ and the bioengineered tooth Boxes indicate the area shown at higher magnification in the centre panels To analyse the structure of the enamel rod and dentin tube the tooth was treated with 40% phosphoric acid for 10 sec and sodium hypochlorite for 15 sec The surface composition of each tooth was analysed by EDX (A) Schematic representation of orthodontic movement of the natural tooth the erupted tooth formed by transplantation of natural tooth germ and the bioengineered tooth (Illustration by R.N.) (B) Oral photograph of the orthodontic appliance designed for the canine jawbone The erupted teeth were continuously loaded with 10 gf of horizontal orthodontic force (from the buccal side to the lingual side) for 30 days using an orthodontic appliance (C) CT images of the tooth movement of the erupted tooth formed by transplantation of natural tooth germ and the bioengineered tooth before orthodontic treatment (left panels blue) and after orthodontic treatment (centre panels Merged images before and after orthodontic treatment are shown (right panels) we demonstrated successful tooth restoration by autologous transplantation of bioengineered tooth germ into a tooth loss region in a postnatal canine model We also determined that the bioengineered tooth erupted into the oral cavity with the features of proper tooth tissue formation and restored physiological tooth function such as the response to orthodontic mechanical force This study represents a substantial advancement in organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future whole-organ regeneration This advancement is significant for the concept of whole-tooth replacement therapy in which a bioengineered tooth germ can be reconstructed utilizing the bioengineered organ germ method and postnatal stem cells This study demonstrates the feasibility of practical tooth replacement therapy by the transplantation of bioengineered tooth germ as an alternative treatment for autologous tooth transplantation we demonstrated that a bioengineered tooth reconstructed from canine permanent tooth germ reproduced the correct tooth structure including calcified components and enamel and dentin microstructure the erupted bioengineered tooth had a single-root shape with the proper periodontal tissue structure and it achieved physiological tooth function in terms of biological response to mechanical stress equivalent to the PDL function of a natural tooth This study shows that transplantation of bioengineered tooth germ has potential as a biological dental treatment that can result in essential functional recovery of lost teeth to satisfy aesthetic and physiological requirements Further studies that can identify tooth-inducible stem cells in elderly patients for the reconstitution of a bioengineered tooth germ are necessary to realize whole-tooth regenerative therapy in the clinic our study demonstrated functional whole-tooth restoration by autologous transplantation of bioengineered tooth germ in a postnatal large-animal model This study represents a significant advancement in organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine This study was designed to demonstrate whether a fully functional tooth bioengineered using postnatal stem cells can be developed in a large-scale animal Tooth germs of mandibular premolar were dissected from 30-day-old beagle dogs to generate the bioengineered tooth germ using our previously reported organ-germ culture method to evaluate whether the bioengineered tooth germ could develop normally subrenal capsule transplantation was performed in immunodeficient mice after two days of organ culture; the mice were analysed histologically 4 canine bioengineered tooth germs were reconstructed using epithelial tissue and mesenchymal single cells derived from permanent premolar tooth germs of 30-day postnatal dogs; the germs were autologously transplanted into the alveolar bone socket in the mandible after two days of organ culture The bioengineered tooth was analysed radiologically by micro-CT histologically by haematoxylin-eosin (HE) and Azan staining and morphologically by scanning electron microscopy an experimental tooth movement model was used to evaluate the proper periodontal ligament function of the bioengineered tooth The study was performed on 6-week-old female immunodeficient mice (Balb/c nu/nu; CLEA Japan) and beagle dogs at 55 days prior to birth and at postnatal day 30 (Toyo-beagle; ORIENTAL YEAST Co. All animals were handled according to protocols and guidelines approved by the animal committee of Okayama University (OKU-2012334 OKU-2012419) and according to the principles of the Declaration of Helsinki Mice were operated on under general anaesthesia induced by intraperitoneal injection of 0.4 mL/kg of 1:1 ketamine hydrochloride (Ketalar 500 mg; Daiichi Sankyo Propharma Co. Japan) and xylazine (Selactar 2% injection; Bayer HealthCare Canines were anesthetized via an intramuscular injection of a mixture of xylazine (8 mg/kg; Bayer HealthCare) and ketamine (80 mg/kg; Daiichi Sankyo Propharma Co. Local anaesthesia with 2% xylocaine containing 1/80,000 epinephrine was additionally provided before bioengineered tooth germ transplantation The canines were kept in single cages with water and nonsolid food These bioengineered tooth germs were cultured on a cell culture insert (0.4 μm pore diameter; BD) in basal medium consisting of α-MEM (Life Technologies 10% foetal bovine serum (FBS; Life Technologies) 100 μM of L-ascorbic acid 2-phosphate (WAKO 100 U/mL of penicillin and 100 μg/mL of streptomycin (SIGMA To evaluate whether the reconstituted tooth germ could develop subrenal capsule transplantation of the natural tooth germ was performed and tooth germs were reconstituted in several combinations into 6-week-old female immunodeficient mice (CLEA) after 2 days of organ culture Four weeks after transplantation in the reconstruction of each tissue and cell combination (i.e. epithelial tissue and mesenchymal cells) and 8 or 12 weeks after transplantation in the reconstruction of each cell and cell combination (i.e. the bioengineered teeth were harvested from the immunodeficient mice and then analysed histologically At 6 months (180 days) after transplantation the erupted natural tooth and bioengineered tooth were harvested from the canine mandible and analysed radiologically To evaluate the function of the periodontal ligament (PDL) several samples of the transplanted natural tooth and the bioengineered tooth were submitted to orthodontic experiments at 180 days after natural or bioengineered tooth germ transplantation when these teeth had erupted into the oral cavity To analyse natural and bioengineered tooth development and experimental tooth movement in the canine mandible computed tomography (CT) images were obtained using a PLANMECA ProMax 3D Max (PLANMECA Finland) and the accompanying analysis software Micro-CT images of the collected bioengineered teeth were obtained using a SkyScan 1174 compact micro-CT (BRUKER CT scans were captured at a resolution of 64 μm in which 269 sections were reconstructed to produce the final images using SkyScan software Collected samples were fixed in 4% paraformaldehyde (PFA) for 3 days and decalcified with formic citric acid for 60 days The paraffin sections were stained with standard haematoxylin and eosin (HE) fixed tissues were embedded in methyl-methacrylate (MMA) resin and undecalcified 30-μm-thick sections were obtained with a micro-cutting machine MMA resin sections were analysed histologically after HE or Azan staining The bioengineered teeth were fixed with 1% formaldehyde and 1% osmium tetroxide for 15 min each Fixed samples were cut longitudinally and treated with 40% phosphoric acid for 10 sec and sodium hypochlorite for 15 sec samples were sputter-coated with osmium plasma and images were obtained using a scanning electron microscope (SEM: S-4800 Type2 The composition of the bioengineered tooth surface was analysed using the energy-dispersive X-ray spectroscopy instrument (EMAX ENERGY EX-350 The orthodontic force was measured by using a tension gauge (Mitutoyo Corporation CT scans were performed to analyse tooth movement using the ProMax 3D CT-machine both before (day 0) and after (day 30) orthodontic treatment Statistical analyses were performed using the chi-square test and p-values less than 0.05 were considered to be statistically significant Analyses were performed using JMP (version 10.0; SAS Institute Inc. 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dentin complexes by subcutaneous implantation of reconstructed human and murine tooth germ elements iPS cells reprogrammed from human mesenchymal-like stem/progenitor cells of dental tissue origin Stem cells in dentistry–part I: stem cell sources Stem cells in dentistry–Part II: Clinical applications Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial Repair of large bone defects with the use of autologous bone marrow stromal cells Direct comparison of autologous and allogeneic transplantation of iPSC-derived neural cells in the brain of a non-human primate The regulation of tooth morphogenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog through epithelial-mesenchymal interactions Gingival fibroblasts as a promising source of induced pluripotent stem cells Role of epithelial-stem cell interactions during dental cell differentiation Differentiation of induced pluripotent stem cells into dental mesenchymal cells Download references This work was supported by the Health and Labour Sciences Research Grants program of the Ministry of Health (Tokyo Medical and Dental University) and Grants-in-Aid for Scientific Researches (A) from MEXT We also received partial support from Organ Technologies Mitsuaki Ono and Masamitsu Oshima: These authors contributed equally to this work Research Institute for Science and Technology Osaka University Graduate School of Dentistry Tohoku University Graduate School of Dentistry Section of Oral Implantology and Regenerative Dental Medicine Graduate School of Tokyo Medical and Dental University This work was partially funded by Organ Technologies Inc This work was performed under the condition of an Invention Agreement between Tokyo University of Science Download citation Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology NKURENKURU- nguuru gomukunda Kavango zoutoke ro Si r rka Ausiku kageve yitundwamo asi yirugana yoyinzi kuna moneka monomvhura murongo edi dakapita morwa nokampani dononzi kwaya paturura nomberewa nye nampili ngoso ruhepo simpe kuna gumu momunene mukunda ogu ‘’ Ame nina hafa mokumutantera asi ministeli zounangesefa nomarandesero GIPF ntani Nored kadikere nokampani dina kuyapaturura nomberewa dawo sinkwa ntani ame kuna kludiworokesa ministeli zoyipanguira nonombunga dimwe depangero ngwendi social security commission vakwameko morwa awo ngoso kuna kusillika vantu kugwana mauwa aga vana wapere kugwana age kahuyungire nonkango edi poopo kahuyungisire mukunda gwendi moutano vana kutumbura asi State of Regional Address (SORA) modoropa zaNkurenkuru noministeli nonombunga dopaumwene kuna kara asi kuna kuhira komatungo aga vatunga vanangesefa womomukunda eyi yina kara asi mulyo unene eyi yina kuwapukurura ekonomi lyetu ,’’ yimo ngoso ana kutanta Kugusako nye eyi yina kara asi kuretaveneeyionohara mukunda gwaKavango zoutokero simpe kuna kara ngwendi nomukunda doponze zodoropa ntani kuna kara moruhepo rorunene age katente asi mukunda kuna kuguvatera momunene kweyi mberewa azi varura yiweka novantu vakaramo zo (NSA) omu zina kuvapa asi ekuliko ngapi lina kugendapo Evango nye lyaNamibia vana kutumbura asi Multidimensionnal Poverty Index (MPI) yitundwamo eyi vatulisirepo mo 2021 makona kono goruhepo monomukunda horowero 14 domoNamibia kwagwene as i ruhepo moKavango kuna kara peguru unene kuhetakanesa sirongo mudima Mukunda kwakara tupu ngwendi nomukunda doponze zodoropa omu muna kara tupu Nkurenkuru asi yizo nye doropa zonene ano Katwitwi kuna kara tupu asi evango lyokutunga kuna kulifatururamomunenenyesimpe kuna kutuguma udigu wononzira erongo lyokupira kuziramo ntani uhaku una pili yiruganeso nayimwe ngos kadi d i l iki re asi posiruwo sosidigu omu lyatugumine unene erongo lipire kuwapa omu yagumione unene ekonomi lyosirongo momunene malikwamo ntani noprojeka kwakere dina sikama momukunda nampili ngoso epangero kuna kukambadara kugusapo udigu wangesi ekuliko momukunda gwetu kapi lina kugendapo moomu tuna yiharere Ose natukara ngorooro tatutumbura maudigu age dogoro ngagagwene ekohonono Nguu ru katente as i mukunda gwatulisapo eyi vana kutumbura asi etjango lyovantu ntani eyi vana pumbwa momunene yipo asi yiva vatere mokutwara ekuliko komeho omu vana hara asi malikwamo gekuliko gahepa kuza nye konomukundahorowerogona oku vana tungu vantu wovanzi Yipo nye asi awo kuna kuhundira mavango ndi nomberewa diruganesa eyi yina kara monomberewa dawo nsene vana hara kugava mauwa nkenye komukunda nampili noprojeka nado dahepa kuruganesa mauzera gangesi Ausiku kattente asi elikwamo lyawo lyekuliko kuna kara asi kudemenena unene konondima morwa evhu eli nye vakara nalyo lyokuvhura kuzangura eyi ono hara ntani kwakara pepi nomukuro kugwedererako nsonso ntani ruha nye rovadinguli ‘’ Eyi kwatunda nye monombapira dekuliko domukunda mokuditumbura mukunda simpe kuna kara nepuro asi nsene vagwana vapunguli wovawa evango olyo vadidilika kuvhura kuretamo erunduruko ntani kuvhura hena kuretesapo yirugana nokugusapo nye ruhepo unene kovadinkantu vetu,’’ yimo ana kutanta ngoso Katima Mulilo- vatungimo modoropa zaOtavi navavakara pepi ngesi kuvhura vakara vana hafa morwa yirugana eyi agava NaTis ngesi vana yireta nye modoropa zawo Vatungimo modoropa zaOtavi kwakara nonyuku dononzi kugenda sinano sosire konodoropa dopeke vakagwane mbatero RA kavakapatwilire mberewa zaNaTis omu navakarugana makona kono gokusinga ntani kugava nombapira dousingi omu navadiwapukurura mberewa ezi kazipaturukire mo 28 Sitarara 2023 Otjiwarongo ndi Grootfontein oku navakagwana mbatero komberewa zaNaTis muna hamene kutjangesa mahauto ntani kugava nombapira pulisiro; kutara ehauto lina wapere monzira kukona kona muntu ana hara kukara musingi ntani kugava nombapira pulisirio dehauto; ntani ehundiro nsene ono hara kutura nomora zopa umwene; ehundiro lyokukwateramo ntani lyosipesiyare eyi yakere asi pwanare ndiro unene mokuyirugana eyi Eyi kuna kara nye asi yihorokwa yanare ngesi NaTis ana reta yirugana nayinye eyi pepi modoropa Mukurona godoropa zaOtavi Isaac !Hoaeb kalikidire ruhafo rwendi nokutanta asi vatungimo ntani vantu womomukunda horowero kapi navagenda hena sinano sosire vaze konodoropa peke vakapapare mbatero koNaTis ‘’Eli kuna kara egwederero omu NaTis anareta sirugana esi pepi novantu Mberewa ezi nazireta mauwa kovantu navenye omu yina kara asi yomutaro pokatji kaRoad Authority nonompitisili edi dinar eta ruhafo Age katente asi vakansela ngesi vana kara nye asi kuna kureta sirugana kovantu nonzira detu ntani elikwamo nye eli lyokuzeresa doropa zetu Mukurona godoropa kahundilire unene vadinkantu asi vagwane nombapira dokulironga kusinga dogoro vakagwane nombapira dene vakare nye vasingi paveta mononzira zetu ‘’Kwato mutungi moOtavi nakara asi gokutara tupu, ogu nahingira alizengure atare vakwawo womoOtavi omu vana kudana. Gwana evango lyoge mombunga ezi, kuna karapo nokomitiye dokulisiga siga dava vatunga moOtavi gavako ndi gwedako nove kweyi wadiva, yimo ana kugwedako ngoso. Age kwahara nkenye apa kuhundira vatungimo moOtavi varuganene kumwe yipo vakulike doropa ezi moukumwe.  anakale@nepc.com.na Ocala-News.com An 87-year-old Silver Springs man was arrested over the weekend after he allegedly pointed a gun at his daughter’s husband while arguing over how the man speaks to her a Marion County Sheriff’s Office corporal responded to a local residence in reference to a domestic incident involving a firearm the corporal made contact with a wheelchair-bound elderly man Onohara allegedly told the corporal that he “had the gun with him” because he was afraid he was going to be attacked He further claimed that the firearm – a .32 caliber revolver – was not loaded and the corporal noted in the report that no bullets were located in the gun The corporal next made contact with Onohara’s son-in-law who is referred to in the report as the victim Onohara was arguing with the victim in the living room when he rolled himself in his wheelchair to another room Onohara soon returned to the living room and reached into his jacket The victim advised that Onohara then pointed the gun at him the victim stated that he “spun (Onohara) around in his wheelchair” and pried the firearm from Onohara’s grasp The victim then allegedly wheeled Onohara out the front door of the residence before leaving him outside and locking the front door until law enforcement arrived on scene Two witnesses spoke with the corporal and corroborated the victim’s statement One of the witnesses advised that Onohara’s finger was “on the trigger” during the incident the witnesses stated that it was placed on the kitchen counter Onohara refused to discuss the incident and requested an attorney Onohara was arrested and transported to Marion County Jail where he is currently being held without bond He is being charged with domestic aggravated assault with a deadly weapon without intent to kill