技术资料

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication

AbstractThe 22q11.2 deletion syndrome (22qDS) is a human disorder where the majority of clinical manifestations originate during embryonic development. 22qDS is caused by a microdeletion in one chromosome 22,including DGCR8,an essential gene for microRNA (miRNA) production. However,the impact of DGCR8 hemizygosity on human development is still unclear. In this study,we generated two human pluripotent cell models containing a single functional DGCR8 allele to elucidate its role in early development. DGCR8+/? human embryonic stem cells (hESCs) showed increased apoptosis as well as self-renewal and differentiation defects in both the naïve and primed states. The expression of primate-specific miRNAs was largely affected,due to impaired miRNA processing and chromatin accessibility. DGCR8+/? hESCs also displayed a pronounced reduction in human endogenous retrovirus class H (HERVH) expression,a primate-specific retroelement essential for pluripotency maintenance. The reintroduction of miRNAs belonging to the primate-specific C19MC cluster as well as the miR-371-3 cluster rescued the defects of DGCR8+/? cells. Mechanistically,downregulation of HERVH by depletion of primate-specific miRNAs was mediated by KLF4. Altogether,we show that DGCR8 is haploinsufficient in humans and that miRNAs and transposable elements may have co-evolved in primates as part of an essential regulatory network to maintain stem cell identity. Graphical Abstract Graphical Abstract View Publication

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome,but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL,4-1BBL,OX40L,and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40,CD25,and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation,revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion. View Publication

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article,we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro,respectively. Furthermore,our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells,along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover,we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion,our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. View Publication

Due to their complex chemical and physical properties,the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2),presence of goblet cells,and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB,n = 9),or two high fiber diets formulated adding 40{\%} corn distillers' dried grains with solubles (DDGS,n = 9) or 30{\%} wheat middlings (WM,n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase,beta-glucanase,mannanase,and galactosidase (n = 8,10,and 9 for CSB+E,DDGS+E and WM+E,respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet,and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general,enzyme supplementation increased gene expression and concentrations of IL-1beta,and reduced the concentrations of IL-4,IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1beta. In accordance with previous studies,treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However,swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1beta. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine,but by mechanisms different to mouse,emphasizing the need for using appropriate models to study responses to dietary fiber in swine. View Publication

Age-related gastrointestinal decline contributes to whole-organism frailty and mortality. Genistein is known to have beneficial effects on age-related diseases,but its precise role in homeostasis of the aging gut remains to be elucidated. Here,wild-type aging mice and Zmpste24-/- progeroid mice were used to investigate the role of genistein in lifespan and homeostasis of the aging gut in mammals. A series of longitudinal,clinically relevant measurements were performed to evaluate the effect of genistein on healthspan. It was found that dietary genistein promoted a healthier and longer life and was associated with a decrease in the levels of systemic inflammatory cytokines in aging mice. Furthermore,dietary genistein ameliorated gut dysfunctions,such as intestinal inflammation,leaky gut,and impaired epithelial regeneration. A distinct genistein-mediated alteration in gut microbiota was observed by increasing Lachnospira abundance and short-chain fatty acid (SCFA) production. Further fecal microbiota transplantation and dirty cage sharing experiments indicated that the gut microbiota from genistein-fed mice rejuvenated the aging gut and extended the lifespan of progeroid mice. It was demonstrated that genistein-associated SCFAs alleviated tumor necrosis factor alpha-induced intestinal organoid damage. Moreover,genistein-associated propionate promoted regulatory T cell-derived interleukin 10 production,which alleviated macrophage-derived inflammation. This study provided the first data,to the authors' knowledge,indicating that dietary genistein modulates homeostasis in the aging gut and extends the healthspan and lifespan of aging mammals. Moreover,the existence of a link between genistein and the gut microbiota provides a rationale for dietary interventions against age-associated frailty. View Publication

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here,we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistochemistry,live imaging,and single-cell transcriptomics. We find that the cytoarchitecture,cell type composition,and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably,however,live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this,the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity,and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. View Publication

We previously demonstrated that leukemia cell lines expressing CD44 and hematopoietic progenitor cells (HPC) from umbilical cord blood (CB) showed rolling on hyaluronic acid (HA)-coated surfaces under physiological shear stress. In the present study,we quantitatively assessed the interaction of HPC derived from CB,mobilized peripheral blood (mPB) and bone marrow (BM) from healthy donors,as well as primary leukemia blasts from PB and BM of patients with acute myeloid leukemia (AML) with HA. We have demonstrated that HPC derived from healthy donors showed relative homogeneous rolling and adhesion to HA. In contrast,highly diverse behavioral patterns were found for leukemia blasts under identical conditions. The monoclonal CD44 antibody (clone BU52) abrogated the shear stress-induced rolling of HPC and leukemia blasts,confirming the significance of CD44 in this context. On the other hand,the immobile adhesion of leukemia blasts to the HA-coated surface was,in some cases,not or incompletely inhibited by BU52. The latter property was associated with non-responsiveness to induction chemotherapy and subsequently poor clinical outcome. View Publication

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However,the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity,i.e.,CD34 and AC133. In addition to differences in surface marker expression,the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast,the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile,the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells. View Publication

ABSTRACTBacteriophages (phages) are emerging as a viable adjunct to antibiotics for the treatment of multidrug‐resistant (MDR) bacterial infections. While intravenous phage therapy has proven successful in many cases,clinical outcomes remain uncertain due to a limited understanding of host response to phages. In this study,we conducted a comprehensive examination of the interaction between clinical‐grade phages used to treat MDR Escherichia coli and Klebsiella pneumoniae infections,and human peripheral blood immune cells. Using whole transcriptome as well as proteomic approaches,we identified a strong inflammatory response to E. coli phage vB_EcoM‐JIPh_Ec70 (herein,JIPh_Ec70) that was absent upon exposure to K. pneumoniae phage JIPh_Kp127. We confirmed that JIPh_Ec70's DNA recognition by the STING pathway was principally responsible for the activation of NF‐kB and the subsequent inflammatory response. We further show that monocytes and neutrophils play a dominant role in phage uptake,primarily through complement‐mediated phagocytosis. Significant differences in complement‐mediated phagocytosis of JIPh_Kp127 and JIPh_Ec70 were observed,suggesting that reduced recognition,phagocytosis,and immunogenicity all contribute to the significantly decreased response to JIPh_Kp127. Our findings contribute to the progress of our understanding of the innate immune response to therapeutic phages and offer potential insights into how to improve the safety and effectiveness of phage therapy. Clinical grade JIPh_Ec70 phages but not JIPh_Kp127 phages elicit a potent inflammatory response in peripheral immune cells. JIPh_Ec70 phagocytic engulfment is facilitated by complement opsonization,resulting in STING activation by phage DNA,driving an inflammatory signaling cascade. Understanding phage immunogenicity will be a key factor in developing effective phage therapies in the coming years. View Publication

Mucosal-associated invariant T (MAIT) cells exhibit different characteristics from those of TCRalpha7.2- conventional T cells. They play important roles in various inflammatory diseases,including rheumatoid arthritis and inflammatory bowel disease. MAIT cells express a single T cell receptor alpha chain,TCRalpha7.2 segment associated with Jalpha33 and CDR3 with fixed length,which recognizes bacteria-derived vitamin B metabolites. However,the characteristics of MAIT cells and TCRalpha7.2+ CD161- T cells have never been compared. Here,we performed RNA sequencing to compare the properties of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. Genome-wide transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells,and TCRalpha7.2+ CD161- T cells were compared and analyzed using causal network analysis. This is the first report comparing the transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. We also identified the predominant signaling pathways of MAIT cells,which differed from those of TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells,through a gene set enrichment test and upstream regulator analysis and identified the genes responsible for the characteristic MAIT cell phenotypes. Our study advances the complete understanding of MAIT biology. View Publication

Single cardiac transmembranous Ca channels have three modes of gating behaviour in the absence of drugs,expressed as current records with brief openings (mode 1),with no openings because of channel unavailability (mode 0 or null mode) and with long-lasting openings and very brief closings that appear only rarely (mode 2). The dihydropyridine Ca agonist Bay K 8644 enhances Ca channel current by promoting mode 2,while the Ca antagonists nitrendipine and nimodipine inhibit the current by favouring mode 0. View Publication