Identification of FAT4 as a positive prognostic biomarker in DLBCL by comprehensive genomic analysis.

The molecular landscapes of diffuse large B-cell lymphoma (DLBCL) remained to be comprehensively investigated with an urgent need to identify novel prognostic biomarkers guiding prognostic stratification and disease monitoring. Baseline tumor samples of 148 DLBCL patients were analyzed using targeted next-generation sequencing (NGS) for mutational profiling, whose clinical reports were retrospectively reviewed. In this cohort, the subgroup of old DLBCL patients (age at diagnosis > 60, N = 80) exhibited significantly higher Eastern Cooperative Oncology Group scores and International Prognostic Index than their young counterparts (age at diagnosis ≤ 60, N = 68). As revealed by the NGS results, PIM1 (43.9%), KMT2D (31.8%), MYD88 (29.7%), and CD79B (27.0%) were identified as the most frequently mutated genes. Aberrations of genes of the immune escape pathway were significantly enriched in the young subgroup, while the altered epigenetic regulators were more abundant in the old patients. FAT4 mutation was identified as a positive prognostic biomarker, associated with longer progression-free survival and overall survival in the entire cohort and the old subgroup, using the Cox regression analyses. However, the prognostic function of FAT4 was not reproduced in the young subgroup. We comprehensively analyzed the pathological and molecular characteristics of old and young DLBCL patients and demonstrated the prognostic value of FAT4 mutation, which requires further validation with sizable cohorts in future research.

Recent progress of theoretical studies on electro- and photo-chemical conversion of CO2 with single-atom catalysts.

The CO2 reduction reaction (CO2RR) into chemical products is a promising and efficient way to combat the global warming issue and greenhouse effect. The viability of the CO2RR critically rests with finding highly active and selective catalysts that can accomplish the desired chemical transformation. Single-atom catalysts (SACs) are ideal in fulfilling this goal due to the well-defined active sites and support-tunable electronic structure, and exhibit enhanced activity and high selectivity for the CO2RR. In this review, we present the recent progress of quantum-theoretical studies on electro- and photo-chemical conversion of CO2 with SACs and frameworks. Various calculated products of CO2RR with SACs have been discussed, including CO, acids, alcohols, hydrocarbons and other organics. Meanwhile, the critical challenges and the pathway towards improving the efficiency of the CO2RR have also been discussed.

Next-Generation Sequencing of Circular RNAs Reveals the Molecular Mechanisms of Chondrogenic Differentiation in Human Adipose-derived Stem Cells.

Adipose-derived stem cells are one of the potential sources of cells for the treatment of cartilage defects. This study aimed to investigate the molecular mechanisms that account for the chondrogenic differentiation of human adipose-derived stem cells (hADSCs). We employed integrin ß1 (ITGB1) overexpression to induce chondrogenic differentiation of hADSCs. Next-generation sequencing was used to determine the mRNAs and circular RNAs (circRNAs) expression profiles in ITGB1-overexpresing and negative control cells. The potential functions of differentially expressed mRNAs were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses. Moreover, differentially expressed circRNAs with the greatest fold change were validated by polymerase chain reaction (PCR), Sanger sequencing, and quantitative real-time PCR (qRT-PCR). These three circRNAs and their downstream microRNAs and mRNAs were used to construct a circRNA-microRNA-mRNA interaction network. The results showed that we identified 713 differentially expressed circRNAs (150 upregulated and 563 downregulated in ITGB1-overexpressing hADSCs versus negative control cells, respectively). Meanwhile, 2383 mRNAs were differentially expressed between two groups (1672 upregulated and 711 downregulated in ITGB1-overexpressing cells compared with the negative control cells). The GO and KEGG analysis results showed that the differentially expressed mRNAs were enriched in biological processes, cellular components, and molecular functions, especially in the phosphatidylinositol 3-kinase (PI3K)-AKT and mitogen-activated protein kinase signaling pathways. Three differentially expressed circRNAs, including hsa_circ_0071127, hsa_circ_0008637, and hsa_circ_0020028, were validated by qRT-PCR. Moreover, the circRNA-microRNA-mRNA network predicted that fibroblast growth factor 2 (FGF2) was a common node regulated by these three circRNAs through several microRNAs, including miR-195-3p, miR-205-3p, and miR-152-3p. We further found that the knockdown of hsa_circ_0020028, but not the two other circRNAs, significantly reduced FGF2 mRNA expression in hADSCs. Furthermore, the knockdown of hsa_circ_0020028 significantly inhibited the protein expression of FGF2, chondrogenic differentiation markers (COL II, aggrecan, and SOX9), and PI3K/AKT signaling in ITGB1-overexpressing hADSCs. This study uncovered the differentially expressed mRNA and circRNA profiles in the chondrogenic differentiation of hADSCs induced by ITGB1 overexpression. Our findings demonstrate that hsa_circ_0020028 regulates the ITGB1 overexpression-mediated chondrogenic differentiation of hADSCs through regulation of FGF2-related signaling pathways.

Rab35 regulates insulin secretion via phogrin in pancreatic ß cells.

Dysfunction of pancreatic ß cell insulin secretion is related to the pathogenesis of type 2 diabetes (T2D). Rab proteins have been shown to be key players in insulin secretion by pancreatic ß cells, and phogrin is a marker for the processes of exocytosis and insulin secretion. The purposes of this study were to clarify the regulatory role of Rab35 in insulin secretion and analyse the Rab35/phogrin interaction mechanism in ß-TC-6 cells. We studied the effects of Rab35 gene overexpression and interference on insulin secretion and phogrin expression and levels in ß-TC-6 cells. The Rab35/phogrin interaction was verified by GST pulldown, co-IP and co-localisation experiments. Here, we report that Rab35 is mainly distributed in the ß-TC-6-cell plasma membrane and cytoplasm. Rab35 overexpression promotes insulin secretion and decreases phogrin expression in ß-TC-6 cells, whereas its silencing significantly inhibits insulin secretion, promotes phogrin expression (p < 0.05) and causes phogrin redistribution. Furthermore, Rab35 silencing suppresses exocytosis of insulin. Rab35 interacts with phogrin, and both proteins co-localise in the plasma membranes and cytoplasm of ß-TC-6 cells. Our study presents novel evidence that Rab35 regulates insulin secretion by inhibiting phogrin expression and causing intracellular phogrin redistribution in pancreatic ß cells.

IL-27 signalling promotes adipocyte thermogenesis and energy expenditure.

Thermogenesis in brown and beige adipose tissue has important roles in maintaining body temperature and countering the development of metabolic disorders such as obesity and type 2 diabetes1,2. Although much is known about commitment and activation of brown and beige adipose tissue, its multiple and abundant immunological factors have not been well characterized3-6. Here we define a critical role of IL-27-IL-27Rα signalling in improving thermogenesis, protecting against diet-induced obesity and ameliorating insulin resistance. Mechanistic studies demonstrate that IL-27 directly targets adipocytes, activating p38 MAPK-PGC-1α signalling and stimulating the production of UCP1. Notably, therapeutic administration of IL-27 ameliorated metabolic morbidities in well-established mouse models of obesity. Consistently, individuals with obesity show significantly decreased levels of serum IL-27, which can be restored after bariatric surgery. Collectively, these findings show that IL-27 has an important role in orchestrating metabolic programs, and is a highly promising target for anti-obesity immunotherapy.

Roux-en-Y Gastric Bypass Improved Insulin Resistance via Alteration of the Human Gut Microbiome and Alleviation of Endotoxemia.

BACKGROUND: Obesity is a main contributing factor for the development of glucose intolerance and type 2 diabetes mellitus (T2D). Roux-en-Y gastric bypass (RYGB) is believed to be one of the most effective treatments to reduce body weight and improve glucose metabolism. In this study, we sought to explore the underlying mechanisms of weight reduction and insulin resistance improvement after RYGB. METHODS: This was a prospective observational study using consecutive samples of 14 obese subjects undergoing bariatric surgery. Main assessments were serum indexes (blood metabolites, glucose-lipid regulating hormones, trimethylamine-N-oxide (TMAO), and lipopolysaccharide-binding protein (LBP), fecal short-chain fatty acids (SCFAs), and gut microbiota. Correlation analysis of the factors changed by RYGB was used to indicate the potential mechanism by which surgery improves insulin resistance. RESULTS: The subjects showed significant improvement on indices of obesity and insulin resistance and a correlated change of gut microbiota components at 1 month, 3 months, and 6 months post-RYGB operation. In particular, the abundance of a counterobese strain, Akkemansia muciniphila, had gradually increased with the postoperative time. Moreover, these changes were negatively correlated to serum levels of LBP and positively correlated to serum TMAO and fecal SCFAs. CONCLUSIONS: Our findings uncovered links between intestinal microbiota alterations, circulating endotoxemia, and insulin resistance. This suggests that the underlying mechanism of protection of the intestine by RYGB in obesity may be through changing the gut microbiota.

Elevated expression of MMP8 and MMP9 contributes to diabetic osteoarthritis progression in a rat model.

BACKGROUND: Knowledge regarding the pathogenesis of osteoarthritis (OA) is very limited. Previous studies have shown that matrix metalloproteinase (MMP) 8 and MMP9 were upregulated in patients with diabetic OA. However, their regulatory functions and mechanisms in diabetic OA are not fully understood. METHODS: Diabetic OA rats were constructed using a high-fat diet combined with streptozotocin (STZ) induction. Safranin O-Fast green staining was used to detect the pathological changes in rat knee cartilage. MMP8 and MMP9 overexpression vectors or siRNAs were injected into diabetic OA rats to overexpress or knockdown the expression of MMP8 and MMP9, which was verified by real-time quantitative PCR (RT-qPCR). The expression of MMP8 and MMP9, chondrocyte differentiation markers collagen type II alpha 1 (COL2A1) and collagen type I alpha 1(COL1A1), and antiapoptotic protein BCL2 were detected using immunohistochemistry (IHC), and the number of apoptotic cells was detected by the transferase-mediated d-UTP nick-end-labeling (TUNEL) assay. RESULTS: High-fat diet combined with STZ-induced rats exhibited joint cartilage damage, morphological changes, and increased expression of MMP8 and MMP9. Overexpression of MMP8 and MMP9 in the joint cavity further aggravated the pathological morphological changes, decreased the expression of COL2A1 and COL1A1, increased the expression of BCL2, and promoted cell apoptosis in diabetic OA rats. The use of siRNA to inhibit MMP8 and MMP9 levels in the cartilage joints significantly reversed the decrease in COL2A1 and COL1A1 expression and partially reversed BCL2 expression and chondrocyte apoptosis. CONCLUSION: MMP8 and MMP9 promoted rat diabetic OA model. The underlying mechanism may be related to inhibiting cartilage differentiation and promoting chondrocyte apoptosis.

ITGB1 promotes the chondrogenic differentiation of human adipose-derived mesenchymal stem cells by activating the ERK signaling.

Adipose-derived mesenchymal stem cell (ADSC) with a high capacity of chondrogenic differentiation was a promising candidate for cartilage defect treatment. This study's objective is to study the roles of integrin ß1 (ITGB1) in regulating ADSC chondrogenic differentiations as well as the underlying mechanisms. The identity of ADSC was confirmed by flow cytometry. ITGB1 gene was overexpressed in human ADSC (hADSC) by transfection with LV003-recombinant plasmids. Gene mRNA and protein levels were examined using quantitative RT-PCR and western blotting, respectively. Differentially expressed mRNAs and proteins were characterized by next-generation RNA sequencing and label-free quantitative proteomics, respectively. ERK signaling and AKT signaling in hADSCs were inhibited by treating with SCH772984 and GSK690693, respectively. ITGB1 gene overexpression substantially increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9) expression but suppressed collagen type I alpha 1 chain (COL1A1) expression in hADSCs. Next-generation RNA sequencing identified a total of 246 genes differentially expressed in hADSCs by ITGB1 overexpression, such as 183 upregulated and 63 downregulated genes. Label-free proteomics characterized 34 proteins differentially expressed in ITGB1-overexpressing hADSCs. Differentially expressed genes and proteins were enriched by different biological processes such as cell adhesion and differentiation and numerous signaling pathways such as the ERK signaling pathway. ERK inhibitor treatment caused substantially enhanced chondrogenic differentiation in ITGB1-overexpressing hADSCs. ITGB1 promoted the chondrogenic differentiation of human ADSCs via the activation of the ERK signaling pathway.

Perforin Acts as an Immune Regulator to Prevent the Progression of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of cirrhosis and major risk factors for hepatocellular carcinoma and liver-related death. Despite substantial clinical and basic research, the pathogenesis of obesity-related NAFLD remains poorly understood. In this study, we show that perforin can act as an immune regulator to prevent the progression of NAFLD. Aged perforin-deficient (Prf-/-) mice have increased lipid accumulation in the liver compared to WT mice. With high-fat diet (HFD) challenge, Prf-/- mice have increased liver weight, more severe liver damage, and increased liver inflammation when compared with WT controls. Mechanistic studies revealed that perforin specifically regulates intrinsic IFN-γ production in CD4 T cells, not CD8 T cells. We found that CD4 T cell depletion reduces liver injury and ameliorates the inflammation and metabolic morbidities in Prf-/- mice. Furthermore, improved liver characteristics in HFD Prf-/- and IFN-γR-/- double knockout mice confirmed that IFN-γ is a key factor for mediating perforin regulation of NAFLD progression. Overall, our findings reveal the important regulatory role perforin plays in the progression of obesity-related NAFLD and highlight novel strategies for treating NAFLD.

Variations in Irisin, Bone Mineral Density, Bone Mineral Content, and Body Composition After Laparoscopic Bariatric Procedures in Obese Adults.

INTRODUCTION: This study was aimed to assess irisin levels in obesity (OB) and T2DM individuals and investigate the dynamic changes of irisin, bone mineral density (BMD), bone mineral content, and body composition in 19 OB patients after laparoscopic bariatric procedures, and also to evaluate the correlation of irisin, with BMD and body composition. METHODS: Forty-five OB, 20 T2DM, and 20 healthy adults had been recruited. Levels of irisin were measured in all subjects. Metabolic characteristics were obtained from OB and T2DM patients. Nineteen patients were randomly assigned to be received Roux-en-Y gastric bypass (LRYGB) or laparoscopic sleeve gastrectomy (LSG) procedure and to be completed 6-month follow-up. Irisin, BMD, bone mineral content, and body composition were measured at each visit. RESULTS: Significantly higher circulating irisin levels were measured in the OB group compared with T2DM and control groups. FINS, C-P, HOMA-IR, FBCI, HBCI, ALT, AST, and UA levels of OB were significantly higher than those of T2DM patients. While FBG and HbA1c of the OB were significantly lower than T2DM group. There were significant differences among circulating irisin, BMD, and body composition after laparoscopic bariatric surgery. Levels of irisin were decreased after operations including both LRYGB and LSG surgery compared with preoperation. At each time point (1, 3, and 6 months) of postoperation, there was no significant difference in percentage of total weight loss between LSG and LRYGB group. The positive correlation of irisin levels with total BMD, muscle, and fat masses were found during 6-month follow-up after surgery. CONCLUSIONS: The levels of irisin were higher in OB patients. There were positive correlations of irisin levels with total BMD, muscle, and fat masses during 6-month follow-up after surgery. Irisin may be involved in the occurrence and development of obese and it is related to BMD and body composition. Both LRYGB and LSG operations could decrease the circulating levels of irisin.

Expression and Significance of MMPs in Synovial Fluid, Serum and PBMC Culture Supernatant Stimulated by LPS in Osteoarthritis Patients With or Without Diabetes.

OBJECTIVE: Patients with Type 2 Diabetes mellitus (T2DM) are prone to osteoarthritis (OA). Matrix metalloproteinases (MMPs), an essential modulator in cartilage matrix homeostasis, increase in T2DM and OA. We aimed to ascertain the expression difference of MMPs and function in mononuclear cells after stimulating by lipopolysaccharide (LPS) in OA patients with or without diabetes. METHODS: 30 knee OA patients without T2DM (OA group), 20 knee OA patients with T2DM (DM-OA group) and 5 healthy volunteers recruited as control were enrolled from January 2016 to January 2017. The expression levels of MMPs in both serum and synovial fluid were initially detected in three groups by enzyme-linked immunosorbent assay (ELISA). After stimulation of peripheral blood mononuclear cell (PBMC) with LPS, the release of MMPs were determined and evaluated. RESULTS: The expression of MMP-1, -7, -8, -9, -10 and -12 in synovial fluid in DM-OA group were significantly higher than in OA group and healthy control. The expression of MMP-1 and -7 in serum were highest in DM-OA group. LPS significantly promotes the production of MMP-1, -8, -9 and -10 in PBMC of each group after 4 h stimulation. It is worth to note that the LPS-stimulated MMP-8 and -9 elevations were more prominent in DM-OA group compared with their counterparts. CONCLUSION: High levels of MMP-1, -7, -8, -9, -10, and -12 in the synovial fluid might be one of important reasons that diabetes patients are more frequently suffered from OA. Inflammation-induced malfunction of mononuclear cells would stimulate MMP-8 and -9 secretion to various extents.

Mitochondrial ROS activate interleukin-1ß expression in allergic rhinitis.

Allergic rhinitis (AR) is the most common cause of inflammation of the nasal mucosa. It is also the most common form of non-infectious rhinitis associated with an immunoglobulin E (IgE)-mediated immune response against allergens. Previous studies have indicated that interleukin-1ß (IL-1ß) has a pathological role in the development of allergic asthma. The present study was designed to assess whether IL-1ß participates in the pathogenesis of AR. A total of 45 patients with AR were enrolled in the present study and were identified to have increased IL-1ß expression expressed by peripheral blood mononuclear cells (PBMCs), and the mitochondrial reactive oxygen species (ROS) and NLRP3 are required for IL-1ß synthesis in monocytes/macrophages and PBMCs from patients with AR. The levels of IL-1ß and interleukin-17 (IL-17) were increased in patients with AR and were positively correlated with each other. The results of the present study suggested that patients with AR have raised mitochondrial ROS levels, which may upregulate the expression of IL-1ß, affecting IL-17-production and serving a role in the pathogenesis of AR.

Elevated RBP4 plasma levels were associated with diabetic retinopathy in type 2 diabetes.

The retinol-binding protein 4 (RBP4) has been postulated to play a role in glucose homeostasis, insulin resistance, and diabetes mellitus in human and animal studies. The aim of the present study was to evaluate the role of RBP4 in Chinese patients with type 2 diabetes mellitus with and without diabetic retinopathy (DR). Plasma RBP4 concentrations were tested in 287 patients with type 2 diabetes. At baseline, demographic and clinical information including presence of DR and vision-threatening DR (VTDR) was collected. The relationship between RBP4 and DR (VTDR) was investigated using logistic regression. Patients with DR or VTDR had significantly higher plasma levels of RBP4 on admission (P<0.0001). Receiver operating characteristics (ROCs) to predict DR and VDTR demonstrated areas under the curve for RBP4 of 0.79 (95% confidence interval (CI): 0.73-0.85) and 0.90 (95% CI: 0.85-0.94), respectively, which were superior to other factors. For each 1 µg/ml increase in plasma level of RBP4, the unadjusted and adjusted risk of DR would be increased by 8% (with the odds ratio (OR) of 1.08 (95% CI: 1.05-1.13), P<0.001) and 5% (1.05 (1.02-1.11), P=0.001), respectively. It was 12% (with the OR of 1.12 (95% CI: 1.07-1.18), P<0.001) and 9% (1.09 (1.05-1.15), P<0.001) for VTDR. The present study shows that elevated plasma levels of RBP4 were associated with DR and VDTR in Chinese patients with type 2 diabetes, suggesting a possible role of RBP4 in the pathogenesis of DR complications. Lowering RBP4 could be a new strategy for treating type 2 diabetes with DR.

Blood glucose and insulin and correlation of SLC25A13 mutations with biochemical changes in NICCD patients.

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a hereditary metabolic disease arising from biallelic mutations of SLC25A13. This study aimed to explore the characteristics of fasting blood glucose (FBG), fasting insulin (FINS) and C-peptide (C-P) levels in NICCD infants, analyze their SLC25A13 genetic mutations and further discuss the correlation between SLC25A13 genetic mutations and biochemical changes. Seventy-two cases of infants with cholestasis disease were gathered. Among them, 36 cases with NICCD diagnosis were case group. Meanwhile, 36 cases with unknown etiology but excluded NICCD were control group. FBG, FINS, C-P, ALT, AST, GGT, ALP, TG, HDL-C, LDL-C and Non-HDL-C were collected from all subjects, and DNA was extracted from venous blood for SLC25A13 mutations detection. The incidence of hypoglycemia was 3% in NICCD group. There were no significant statistical difference of FBG, FINS and C-P between NICCD and INC groups ( P > 0.05). ALT, LDL-C and Non-HDL-C levels in NICCD group were lower than the INC group, while SLC25A13 mutations were associated with the level of GGT ( P < 0.05). Ten different SLC25A13 genetic mutations were detected, among which, 851del4, IVS16ins3kb, IVS6+5 G > A and 1638ins23 mutations made up 82% of all mutations. The incidence of hypoglycemia may be higher in small gestational age infants with NICCD. Low LDL-C may be one of the characteristics of dyslipidemia in NICCD infants. There was a correlation between SLC25A13 gene mutations distribution and the GGT level, but the meaning of this finding remains to be further in-depth study. Impact statement This study aims to compare FBG, FINS, C-P, other biochemical and clinical manifestations between NICCD and non-NICCD infants, and discuss differential diagnosis of NICCD and INC beyond the genetic analysis. And investigate the correlation between SLC25A13 genetic mutations and biochemical changes. This work presented that incidence of hypoglycemia may be higher in small gestational age infants with NICCD. Low LDL-C may be one of the characteristics of dyslipidemia in NICCD infants. There was a correlation between SLC25A13 gene mutations distribution and the GGT level.

Functional conservation study of polarity protein Crumbs intracellular domain.

The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.

In situ single molecule detection of insulin receptors on erythrocytes from a type 1 diabetes ketoacidosis patient by atomic force microscopy.

Type 1 diabetes is an insulin-dependent metabolic disorder always associated with ketoacidosis and a high morbidity rate in teenagers. The in situ single molecule detection of insulin receptors on healthy and diseased erythrocytes is helpful to understand the pathomechanism of type 1 diabetes ketoacidosis (T1-DKA), which would also benefit the diagnosis and treatment of T1-DKA. Here, we demonstrated, for the first time, the single molecule interaction between insulin and insulin receptor on erythrocytes from a healthy volunteer and a T1-DKA patient using high sensitivity atomic force microscopy (AFM) in PBS solution. The single molecule force results demonstrated the decreased binding force and binding probability between insulin and insulin receptor on T1-DKA erythrocytes, implying the deficit of insulin receptor functions in T1-DKA. The binding kinetic parameters calculated from dynamic force spectroscopy indicated that the insulin-insulin receptor complexes on T1-DKA erythrocytes were less stable than those from healthy volunteer. Using high resolution AFM imaging, a decreased roughness was found both in intact T1-DKA erythrocytes and in the purified membrane of T1-DKA erythrocytes, and an increased stiffness was also found in T1-DKA erythrocytes. Moreover, AFM, which was used to investigate the single molecule interactions between insulin-insulin receptor, cell surface ultrastructure and stiffness in healthy and diseased erythrocytes, was expected to develop into a potential nanotool for pathomechanism studies of clinical samples at the nanoscale.

Wnt/ß-catenin signaling may be involved with the maturation, but not the differentiation, of insulin-producing cells.

Wnt/ß-catenin signaling (WNT) has widespread roles during stem cell differentiation. Whether WNT suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of WNT signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of WNT were dynamically regulated in a 12-day IPC differentiation assay. Specifically, protein levels of Wnt1, ß-catenin, and GSK3ß steadily increased from day 0 to day 9 and rapidly decreased by day 12 of differentiation. Similarly, endonuclear ß-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two WNT pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that WNT signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of WNT signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of WNT signaling. Next, we examined the effect of maintaining active WNT signaling from day 9 to day 12 of IPC differentiation. Differentiating cells were treated with Wnt1 on day 9, when WNT signaling is typically turned off, and subjected to gene expression analysis on day 12. Remarkably, Wnt1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that WNT may not be necessary for IPC differentiation but may be involved in IPC maturation.

Inactivation of Wnt/ß-catenin signaling in human adipose-derived stem cells is necessary for chondrogenic differentiation and maintenance.

The Wnt/ß-catenin signaling pathway plays critical roles in self-renewal and differentiation of mesenchymal stem cells. However, very little is known about its role in the chondrogenesis of human adipose-derived stem cells (hADSCs). In this study, we analyzed protein expression of several key components of the Wnt/ß-catenin signaling pathway using a 21-day in vitro model of hADSC chondrogenesis. Wnt1, ß-catenin, and GSK3ß levels increased sharply at day 12, peaked at day 18, and then declined. Expression of TCF1, a target gene of Wnt/ß-catenin signaling, closely followed that of Wnt1. These results were consistent with changes in endonuclear ß-catenin levels. Gene expression of the chondrocyte-specific markers, collagen type II (COL II), SOX9, and aggrecan, increased during hADSC chondrogenesis, peaked at day 12, and then declined. Adding a Wnt inhibitor (days 0-21) resulted in consistently elevated levels of COL II, SOX9, and aggrecan mRNA. In contrast, adding Wnt1 (days 0-21) to cultures led to sustained Wnt/ß-catenin signaling over the 21 days and suppressed expression of chondrocyte-specific markers. Moreover, adding Wnt1 at late stages of differentiation (day 18) further diminished chondrocyte-specific marker expression. Together, these results showed that inactivation of Wnt/ß-catenin signaling is needed for the progression of chondrogenesis and the maturation and phenotype maintenance of chondroid cells.

GLP-1 could improve the similarity of IPCs and pancreatic beta cells in cellular ultrastructure and function.

Transplantation of functional insulin-producing cells (IPCs) provides a novel mode for insulin replacement, but is often accompanied by many undesirable side effects. Our previous studies suggested that IPCs could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. To obtain a better method through which to acquire more similar IPCs, we compared the difference between IPCs of the GLP-1 group and IPCs of the non-GLP-1 group in the morphological features in cellular level and physiological function. The levels of insulin secretion were measured by ELISA. The insulin and glucagon-like peptide-1 (GLP-1) mRNA gene expression was determined by real-time quantitative PCR. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy (LCSM). Intracellular Ca(2+) levels and Glucagon-like peptide-1 receptor (GLP-1R) levels were determined by flow cytometer (FCM). We found that IPCs of the GLP-1 group had bigger membrane particle size and average roughness (Ra ) than IPCs of the non-GLP-1 group but still smaller than normal human pancreatic beta cells. The physiology function of IPCs of the GLP-1 group were much closer to normal human pancreatic beta cells than IPCs of the non-GLP-1 group. GLP-1 could improve the similarity of IPCs from human adipose tissue-derived mesenchymal stem cells and pancreatic beta cells in cellular ultrastructure and function.