Yokohama City University

A high expression of the enzyme beta1,4-galactosyltransferase-3, or B4GALT3 is associated with noticeably shortened survival rates in several types of immunotherapy cancersdeficiency in mice TIME inhibits tumor growth. The study shows that a significant reduction of glycosylation -- a type of protein modification -- on T cell surfaces correlates with increases in CD8+ immune cells infiltrating tumors. Weakly immunogenic and strongly immunogenic tumor cells were subcutaneously transplanted  into B4GALT3 knockout and wild-type mice, to examine for tumor cell growth. Only the knockout mice suppressed the growth of strongly immunogenic tumor cells. An old campaign slogan for cough syrup, "It tastes awful. And it works," seemed to imply that any sweet content might have diminished the medicinal effect. Sweetness, in the case of cancer, appears as a chain of sugar molecules attached to proteins by beta1,4-galactosyltransferase-3, or B4GALT3 According to the Cancer Genome Atlas, a high expression of this enzyme is associated with noticeably shortened survival rates in several types of immunotherapy cancers, such as neuroblastoma, cervical, and bladder cancer. However, the specific role of B4GALT3 in the tumor immune microenvironment -- or TIME -- was still unknown. Now, a team of researchers at Kyoto University and Yokohama City University has found that B4GALT3 deficiency in mice TIME inhibits tumor growth. The study shows that a significant reduction of glycosylation -- a type of protein modification -- on T cell surfaces correlates with increases in CD8+ immune cells infiltrating tumors. "In B4GALT3 knockout or KO mice, we demonstrated the potential of manipulating glycosylation of the T cell surface as a new approach to cancer immunotherapy," says Heng Wei of Kyoto University's Graduate School of Medicine. By purifying membrane proteins and enzymatically cleaving them to enrich glycopeptides, the team could identify the sites and structures of glycans -- complex and highly branched sugar chains -- and the amount of glycoproteins. The role of glycans has attracted much attention in studies on cancer cells, which proliferate and metastasize, depending on their interaction with their microenvironment. The team subcutaneously transplanted weakly immunogenic and strongly immunogenic tumor cells into B4GALT3 knockout and wild-type mice, to examine for tumor cell growth. Only the knockout mice suppressed the growth of strongly immunogenic tumor cells. In addition, the increased CD8+ T cells in knockout mice secreted anti-cancer compounds Interferon-γ and Granzyme B. "We found that the loss of B4GALT3 caused significant fluctuations in gene expression in the immune system, a discovery which has significantly changed the direction of our next phase of research," adds coauthor Chie Naruse. "We have gained insight into the role of glycans in cancer progression and immune response, inspiring possibilities of B4GALT3-centered cancer therapies," says team leader Masahide Asano.

The crocodilian edition of hemoglobins -- the scuba tanks of the blood -- work so well that crocs can go hours without air. The hyper-efficiency of that adaptation has led some biologists to wonder why, of all the jawed vertebrates, crocodilians were the lone group to hit on such an optimal solution to making the most of a breath. After resurrecting the hemoglobin of ancient crocodilian ancestors, a team may have an answer. It can pogo-stick along at 50-plus miles per hour, leaping 30-odd feet in a single bound. But that platinum-medal athleticism falls by the wayside at a sub-Saharan riverside, the source of life and death for the skittish impala stilling itself for a drink in 100-degree heat. A Nile crocodile has silently baptized itself in that same muddy river for the past hour. When the unseen apex predator lashes from the water to seize the impala, its infamous teeth latch onto a hindquarter, jaws clenching with 5,000 pounds of force. Yet it's the water itself that does the killing, with the deep-breathed reptile dragging its prey to the deep end to drown. The success of the croc's ambush lies in the nanoscopic scuba tanks -- hemoglobins -- that course through its bloodstream, unloading oxygen from lungs to tissues at a slow but steady clip that allows it to go hours without air. The hyper-efficiency of that specialized hemoglobin has led some biologists to wonder why, of all the jawed vertebrates in all the world, crocodilians were the lone group to hit on such an optimal solution to making the most of a breath. By statistically reconstructing and experimentally resurrecting the hemoglobin of an archosaur, the 240-million-year-old ancestor of all crocodilians and birds, the University of Nebraska-Lincoln's Jay Storz and colleagues have gleaned new insights into that why. Rather than requiring just a few key mutations, as earlier research suggested, the unique properties of crocodilian hemoglobin stemmed from 21 interconnected mutations that litter the intricate component of red blood cells. That complexity, and the multiple knock-on effects that any one mutation can induce in hemoglobin, may have forged an evolutionary path so labyrinthine that nature failed to retrace it even over tens of millions of years, the researchers said. "If it was such an easy trick -- if it was that easy to do, just making a few changes -- everyone would be doing it," said Storz, a senior author of the study and Willa Cather Professor of biological sciences at Nebraska. All hemoglobin binds with oxygen in the lungs before swimming the bloodstream and eventually releasing that oxygen to the tissues that depend on it. In most vertebrates, hemoglobin's affinity for capturing and holding oxygen is dictated largely by molecules known as organic phosphates, which, by attaching themselves to the hemoglobin, can coax it into releasing its precious cargo. But in crocodilians -- crocodiles, alligators and their kin -- the role of organic phosphates was supplanted by a molecule, bicarbonate, that is produced from the breakdown of carbon dioxide. Because hardworking tissues produce lots of carbon dioxide, they also indirectly generate lots of bicarbonate, which in turn encourages hemoglobin to dispense its oxygen to the tissues most in need of it. "It's a super-efficient system that provides a kind of slow-release mechanism that allows crocodilians to efficiently exploit their onboard oxygen stores," Storz said. "It's part of the reason they're able to stay underwater for so long." As postdoctoral researchers in Storz's lab, Chandrasekhar Natarajan, Tony Signore and Naim Bautista had already helped decipher the workings of the crocodilian hemoglobin. Alongside colleagues from Denmark, Canada, the United States and Japan, Storz's team decided to embark on a multidisciplinary study of how the oxygen-ferrying marvel came to be. Prior efforts to understand its evolution involved incorporating known mutations into human hemoglobin and looking for any functional changes, which were usually scant. Recent findings from his own lab had convinced Storz that the approach was flawed. There were plenty of differences, after all, between human hemoglobin and that of the ancient reptilian creatures from which modern-day crocodilians evolved. "What's important is to understand the effects of mutations on the genetic background in which they actually evolved, which means making vertical comparisons between ancestral and descendant proteins, rather than horizontal comparisons between proteins of contemporary species," Storz said. "By using that approach, you can figure out what actually happened." So, with the help of biochemical principles and statistics, the team set out to reconstruct hemoglobin blueprints from three sources: the 240-million-year-old archosaur ancestor; the last common ancestor of all birds; and the 80-million-year-old shared ancestor of contemporary crocodilians. After putting all three of the resurrected hemoglobins through their paces in the lab, the team confirmed that only the hemoglobin of the direct crocodilian ancestor lacked phosphate binding and boasted bicarbonate sensitivity. Comparing the hemoglobin blueprints of the archosaur and crocodilian ancestors also helped identify changes in amino acids -- essentially the joints of the hemoglobin skeleton -- that may have proved important. To test those mutations, Storz and his colleagues began introducing certain croc-specific mutations into the ancestral archosaur hemoglobin. By identifying the mutations that made archosaur hemoglobin behave more like that of a modern-day crocodilian, the team pieced together the changes responsible for those unique, croc-specific properties. Counter to conventional wisdom, Storz and his colleagues discovered that evolved changes in hemoglobin's responsiveness to bicarbonate and phosphates were driven by different sets of mutations, so that the gain of one mechanism was not dependent on the loss of the other. Their comparison also revealed that, though a few mutations were enough to subtract the phosphate-binding sites, multiple others were needed to eliminate phosphate sensitivity all together. In much the same way, two mutations seemed to directly drive the emergence of bicarbonate sensitivity -- but only when combined with or preceded by other, easy-to-miss mutations in remote regions of the hemoglobin. Storz said the findings speak to the fact that a combination of mutations might yield functional changes that transcend the sum of their individual effects. A mutation that produces no functional effect on its own might, in any number of ways, open a path to other mutations with clear, direct consequences. In the same vein, he said, those later mutations might influence little without the proper stage-setting predecessors already in place. And all of those factors can be supercharged or waylaid by the environment in which they unfold. "When you have these complex interactions, it suggests that certain evolutionary solutions are only accessible from certain ancestral starting points," Storz said. "With the ancestral archosaur hemoglobin, you have a genetic background that makes it possible to evolve the unique properties that we see in hemoglobins of modern-day crocodilians. By contrast, with the ancestor of mammals as a starting point, it may be that there's some way that you could evolve the same property, but it would have to be through a completely different molecular mechanism, because you're working within a completely different structural context." For better or worse, Storz said, the study also helps explain the difficulty of engineering a human hemoglobin that can mimic and approach the performance of the crocodilian. "We can't just say, 'OK, it's mainly due to these five mutations. If we take human hemoglobin and just introduce those mutations, voilà, we'll have one with those same exact properties, and we'll be able to stay underwater for two hours, too,'" Storz said. "It turns out that's not the case. "There are lots of can't-get-there-from-here problems in the tree of life." Storz, Natarajan and Bautista conducted the study with Signore, now at the University of Manitoba; Aarhus University's Angela Fago; Mississippi State University's Federico Hoffmann; and Yokohama City University's Jeremy Tame. The researchers detailed their findings in the journal Current Biology, receiving support from the National Science Foundation and National Institutes of Health.

Novel Organoid Study at Cincinnati Children's Exposes Double-Agent Role of Puzzling Gene Variant CINCINNATI, Oct. 13, 2022 /PRNewswire/ -- One the many unhealthy outcomes of America's state of obesity occurs when people start building up excess fat around their liver, a condition called non-alcoholic fatty liver disease (NAFLD). Obesity affects 42% of Americans. That high obesity rate is a major reason why an estimated 24% of Americans have developed NAFLD. Obesity also contributes strongly to the approximately 10% of Americans who have type 2 diabetes. Continue Reading Diabetes NASH study from Cincinnati Children's demonstrates the value organoid technology offers for medical research. Tweet this When type 2 diabetes and fatty liver disease occur together a single gene variant—GCKR-rs1260326 TT—can sharply accelerate fat accumulation as shown by the green-stained areas of the liver organoid shown in the lower right panel. Study led by experts at Cincinnati Children's. Source: Cell. When people have both type 2 diabetes and NAFLD, they can have a sharply elevated risk of developing nonalcoholic steatohepatitis (NASH), a condition that goes beyond fatty liver to cause life-threatening liver inflammation and scarring. NASH also can trigger liver cancer. However, this path toward severe liver damage is not automatic. Most people who have NAFLD never develop NASH, not even if they also have type 2 diabetes. Scientists have long suspected that genetic factors must play a role, but so far, few risk variants have been made clear. Now a study led by experts at Cincinnati Children's and two research centers in Japan has tapped into the emerging power of organoid medicine to fill in some of the missing pieces. Their findings, which involved a novel method of producing liver organoids from multiple cell donors, may ultimately help doctors predict and treat the people most at-risk of NAFLD leading to severe complications. The study details were published online Oct. 13, 2022, in the journal Cell. Learning from large numbers of human liver organoids This work was led by principal investigator Takanori Takebe, MD, PhD, who holds research appointments in both Cincinnati and Japan. In recent years, Takebe's team has demonstrated the ability to mass produce human liver organoids. These tiny-but-functional mini organs are grown in the lab from human stem cells and can be customized to reflect specific disease conditions. This study is among the earliest applications of the new organoid production technology for studying the causes of human disease at personalized and population level. The outcome: human data rather than animal data that reflects many—but not all—of the results researchers might learn from a clinical trial. In this case, the research team grew liver organoids from the cells of 24 donors. To validate their usefulness as models for the disease, the behavior of these organoids were compared to data from more than 1,000 people with biopsy-proven NASH. (Watch a video abstract describing the process) The team's experiments ultimately revealed that a single gene variant—GCKR-rs1260326 TT—acts as a dangerous double agent. When the liver is functioning in diabetic conditions (high glucose and high insulin levels), the presence of this variant sharply accelerates NAFLD fat accumulation. But when a liver is working within a healthy blood-sugar balance, that same variant actually helps prevent liver scarring more effectively than NASH livers that carry the normal gene. "It is very exciting to be able to recapitulate genetically-determined individual's phenotype in an organoid model," says first author Masaki Kimura, PhD. " Our series of experiments using data from organoids and patients are further leveraged to disentangle the puzzling role of GCKR-rs1260326, revealing this variant exerts NASH protection and promotion depending on diabetes complications." Implications for clinical care The data from functioning human livers rapidly raised implications for clinical care. For example, the team reports that metformin—a widely prescribed diabetes treatment—made liver organoid function even worse when the gene variant was present. However, replacing metformin with a combination of two drugs, nicotinamide riboside (NR) and nitazoxanide (NTZ), appears to stabilize the organoids' function. This unexpected finding traces to changes the gene variant appears to cause in the liver cells' energy consumption rates. Metformin appears to further slow already weakened metabolic rates that occur when diabetes and NAFLD are combined. In fact, the team matched this effect to HbA1c test results, which are routinely used to measure a person's overall diabetes control. When the variant was present—and HbA1c values were within normal <5.7% ranges—several scores of liver function were better than those without the variant. In stark contrast, when HbA1c values were in the diabetic >6.4% range, the scores were much worse for the cohort carrying the variant. "We found that en masse population-based analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH) factors, opening up a door for an array of future personalized medicine applications propelled by human organoids," Takebe says. "We also found, in collaboration with industry collaborators, that drugs that can uncouple oxidative mechanisms within cells helped improve mitochondrial function and may help livers adapt to the presence of increased fat. This suggests new potential ways to manage diabetic NASH." About the study In addition to Takebe and Kimura, co-authors from Cincinnati Children's included Takuma Iguchi, PhD, Kentaro Iwasawa, MD, PhD, Andrew Dunn, PhD, Wendy Thompson, PhD, Praneet Chaturvedi, MS, Aaron Zorn, PhD, Michelle Wintzinger, BS, Mattia Quattrocelli, PhD, Miki Watanabe-Chailland, PhD, Gaohui Zhu, MD, PhD, Masanobu Fujimoto, MD, PhD, Meenasri Kumbaji, MS, and Vivian Hwa, PhD. The study included contributors from Tokyo Medical and Dental University, Yokohama City University, Gilead Sciences and The Liver Company, Inc. This study was supported by funds from the National Institutes of Health, the Japan Agency for Medical Research and Development, the Falk Transformational Awards Program, JST Moonshot R&D, the Takeda Science Foundation award, the Mitsubishi Foundation, and the Cincinnati Children's Research Foundation. SOURCE Cincinnati Children's Hospital Medical Center