Cell-specific Factor Research Articles

Momordicine-I Suppresses Head and Neck Cancer Growth by Reprogrammimg Immunosuppressive Effect of the Tumor-Infiltrating Macrophages and B Lymphocytes.

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy.

Thrombin Priming Promotes the Neuroprotective Effects of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Via the HGF/AKT/STAT3 Signaling Pathway.

Mesenchymal stem cells (MSCs) directly differentiate into neurons and endothelial cells after transplantation, and their secretome has considerable potential for treating brain injuries. Previous studies have suggested that the effects of MSCs priming with exposure to hypoxia, cytokines, growth factors, or chemical agents could optimize the paracrine potency and therapeutic potential of MSCs. Studies have suggested that thrombin-primed Wharton's Jelly-derived mesenchymal stem cells (Th.WJ-MSCs) significantly enhance the neuroprotective beneficial effects of naive MSCs in brain injury such as hypoxic-ischemic brain injury (HIE) and intraventricular hemorrhage (IVH). This study aimed to characterize WJ-MSCs in terms of stem cell markers, differentiation, cell proliferation, and paracrine factors by comparing naive and Th.WJ-MSCs. We demonstrated that compared with naive MSCs, Th.MSCs significantly enhanced the neuroprotective effects in vitro. Moreover, we identified differentially expressed proteins in the conditioned media of naive and Th.WJ-MSCs by liquid chromatography-tandem mass spectrometry analysis. Secretome analysis of the conditioned medium of WJ-MSCs revealed that such neuroprotective effects were mediated by paracrine effects with secretomes of Th.WJ-MSCs, and hepatocyte growth factor was identified as a key paracrine mediator. These results can be applied further in the preclinical and clinical development of effective and safe cell therapeutics for brain injuries such as HIE and IVH.

Angiotensin-(1-7) Improves Islet β-cell Dedifferentiation by Activating PI3K/Akt/FoxO1 Pathway.

Islet β-cell dedifferentiation may be the main cause of reduced insulin secretion. Angiotensin-(1-7) [Ang-(1-7)] can attenuate high glucose-induced apoptosis and dedifferentiation of pancreatic β-cell, but the specific signal transduction pathway and mechanism are not yet clear. This study aimed to investigate the effects of Ang-(1-7) on high glucose-induced islet β-cell dedifferentiation by activating the phosphatidylinositol-3-kinase/Protein kinase B/ Forkhead box transcription factor O1 (PI3K/Akt/FoxO1) signaling pathway. The mouse islet β-cell line MIN6 cells were passaged and cultured and randomly divided into five groups: control (Con) group, high glucose (HG) group, HG with Ang-(1-7) group, HG with Ang-(1-7) and specific MasR antagonist A-779 group, and HG with Ang-(1-7) and PI3K inhibitor LY294002 group. After 48 hours, glucose-stimulated insulin secretion (GSIS) was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of β-cell-specific factors (Pancreatic duodenal homeobox-1 (Pdx1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A(MafA)) and endocrine progenitor cell-specific factors (Octamer binding transcription factor 4(Oct4), Nanog) were measured by Real Time-PCR and Western blot. The factors of protein expression levels of PI3K/Akt/FoxO1 signaling pathway (Akt, p-Akt, Fox- O1, p-FoxO1) were determined by Western blot. We observed for the first time that high glucotoxicity can induce dedifferentiation of pancreatic islet β-cell, causing a decrease in insulin secretion levels and expression of Pdx1, MafA, p-- FoxO1, and p-Akt and an increase in expression of Oct4 and Nanog. After Ang-(1-7) intervention, insulin secretion levels and expression of Pdx1, MafA, p-FoxO1 and p-Akt were increased, and the levels of Oct4 and Nanog were reduced. However, A-779 and LY294002 could reverse this effect. During these processes, the total Akt and total FoxO1 expression did not change significantly. Ang-(1-7) may prevent high glucose-induced pathological dedifferentiation of pancreatic β-cell by activating the PI3K/Akt/FoxO1 signaling pathway.

A Novel Approach to Epigenetic Profiling in Acute Myeloid Leukemia: Mapping Methylation and Hydroxymethylation in Genomic and Cell-Free DNA

Introduction: Acute Myeloid Leukemia (AML) is marked by uncontrolled growth of undifferentiated myeloid cells. DNA methylation patterns in AML have been thoroughly researched, however, studies on hydroxy methylation patterns, indicating active DNA methylation erasure, are still evolving. Our principal goal was to detect differential methylation and hydroxy methylation regions between AML and non-malignant samples. We proposed that simultaneous profiling of blood cells and circulating cell-free DNA would offer more insights into the disease mechanism. Therefore, we combined these data sources to provide a comprehensive epigenetic picture, assuming that this dual approach could reveal new AML biomarkers and enhance our understanding regarding AML pathobiology. Methods: We collected peripheral blood samples from 16 newly diagnosed AML patients (median age 64, age range 36-85) treated at Tel Aviv Sourasky Medical Center, Israel, and 33 age-matched healthy volunteers (NCT05735704). We utilized novel microarray technology to examine methylation and hydroxy methylation patterns in both genomic and cell-free DNA. Following differential methylation region extraction, we used DAVID functional annotation tool to interpret our results' biological significance. Results: This report presents the preliminary methylation data results. Methylation analysis unveiled 474 and 475 differentially methylated regions between AML and controls in genomic and cell-free DNA analysis, respectively, with only 18 common regions. The clear AML-control distinction is displayed in hierarchical clustering heatmaps (Figure 1a, 1b). Further DAVID tool analysis revealed enrichment in biological processes such as aging, cell cycle regulation, cell morphogenesis, and MAP kinase activity - processes closely linked with AML. Notably, genomic data analysis showed significant enrichment of the ‘transcriptional misregulation in cancer’ pathway, involving genes encoding vital transcription factors for cell development and differentiation. Figure 2 demonstrates the biological pathways for genomic and cell free data analysis. Conclusions: This research underscores the importance of combined epigenetic data from malignant tissue and its microenvironment, obtained by examining genomic and cell-free DNA. Both datasets exhibit striking differences between AML and control samples. We detected enrichment of AML-related biological pathways within differential regions. Our findings of enriched biological pathways align with existing literature, reinforce our analysis validity, and support our combined view of methylation patterns in genomic and cell-free DNA. By suggesting a combined view of these two types of DNA, we provide a more comprehensive perspective on AML's complex epigenetic landscape. It underscores the value of such integrative epigenetic profiling in shedding new light on the intricacies of AML development and progression, which may eventually guide the discovery of novel therapeutic targets.

In Vitro Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Diacerein.

Tissue engineering is the application system that tries to restore damaged tissues by different approaches, such as cellular therapy, application of cell differential factors, and various materials. One of the important goals in tissue engineering is to guide stem cells directly to the desired tissue, and researchers tried to utilize different molecules as effective factors to improve this technique. This study aims to demonstrate the effects of diacerein, a slow-acting drug for the treatment of osteoarthritis, on mesenchymal stem cell proliferation and evaluate its potential in the chondrogenesis process. Stem cells were isolated from adipose tissue, characterized by flow cytometry, and cells were treated with 10-5M diacerein for three weeks. Chondrogenic gene expression of SOX9, COL2A1, ACAN, and TGFB1 were analyzed by qRT-PCR and immunocytochemistry techniques. Our results showed that diacerein increased the expression of the following genes involved in chondrogenesis: SOX9 (2.9-fold, P < 0.00), COL2A1 (2.2-fold, P < 0.00), ACAN (2.7-fold, P < 0.00), and TGFB1 (2.6-fold, P < 0.00). Immunocytochemistry results also showed increased production of collagen type II as the main protein marker for chondrocytes. We observed that diacerein alone could initiate and enhance chondrogenesis, and it can be used as a differentiation factor for stem cells to chondrocyte besides its ability to inhibit IL-1β. Knowing the actual function of diacerein, it could be a good candidate for the treatment of osteoarthritis.

A combined bioinformatics and experimental approach identifies RMI2 as a Wnt/β-catenin signaling target gene related to hepatocellular carcinoma

BackgroundThe Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. There are several target genes known to be key players in tumorigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important to understanding Wnt/β-catenin-mediated carcinogenesis. In this study, we developed a combined bioinformatics and experimental approach to find potential target genes.MethodsLuciferase reporter assay was used to analyze the promoter activity of RMI2. WST1 cell proliferation assays and transwell assays were performed to determine the proliferation and migration capacities of RMI2 overexpressing or knockdown stable hepatic cells. Finally, xenograft experiments were performed to measure the tumor formation capacity in vivo.ResultsThe results showed that RMI2 mRNA was upregulated after LiCl treatment and Wnt3a-conditioned medium in a culture of SK-hep-1 cell lines. A chromatin immunoprecipitation (ChIP) assay showed that the β-catenin/T cell-specific factor (TCF) complex binds to the putative TCF binding site of the RMI2 promoter. We then found a TCF binding site at − 333/− 326 of the RMI2 promoter, which is crucial for β-catenin responsiveness in liver cell lines. RMI2 was overexpressed in hepatoma tissue and cell lines, and it promoted the migration and invasion of HCC cells. Moreover, RMI2 upregulated the expression of epithelial-mesenchymal transition (EMT) markers and the Wnt3a/β-catenin-related genes, but silencing RMI2 had the opposite effects. Notably, the expression of RMI2 was positively correlated with the clinical data of HCC patients who had significantly shorter overall survival (OS) and disease-free survival (DFS) (Both: P < 0.05). In addition, a total of 373 HCC patients’ data from the Caner Genome Atlas project (TCGA) were used to validate our findings.ConclusionsTaking all these findings together, we determined that RMI2 was a new target gene of the Wnt/β-catenin signaling pathway. We also found that RMI2 promotes EMT markers, HCC cell invasion, and metastasis, which indicated that RMI2 is a potential target for preventing or at least mitigating the progression of HCC.

THU151 Undiagnosed MODY In Case Of Morbid Obesity

Abstract Disclosure: A. Abdulkader: None. S.B. Ten: None. MODY is suspected in nonobese subjects with hyperglycemia, negative islet cell antibodies, and a family history of diabetes. Children with morbid obesity and diabetes can still have MODY and require different treatment. Case report The patient was born after a pregnancy complicated with gestational diabetes, treated with insulin, BW 9 1/2 lbs. He had early onset obesity from 3 yrs. of age. At 8 yrs., he developed impaired glucose tolerance, elevated triglycerides, low HDL, and fatty liver. He developed diabetes at 11 yrs. and was treated with Insulin, but treatment was not effective, Hb A1c was 11.4%, and BMI was 51.8 kg/m2. At 12 yrs., he was diagnosed with MODY on the base of normal C-peptide, negative islet cell AB, strong family history of diabetes: mother, uncle and MGM had diabetes. Insulin was discontinued. Glyburide and Metformin were started that improved his glucose level significantly. Hb A1c improved to 6.2%. Diet was changed, physical exercises were added, and he lost 25 lbs., BMI improved to 44.8 kg/m2. His mother, 46 yrs. old, was diagnosed with diabetes type 1 at 18 yrs. and treated with Insulin. She also had morbid obesity with BMI 48 kg/m2. She developed diabetic neuropathy, nephropathy, and had amputation of the left leg below the knee, eGFR 44 mL/min/1.73m2, Hb A1c 10.5%. She was diagnosed with MODY on the base of normal C-peptide 3.21 ng/mL, negative islet cell AB, family history of diabetes: her mother, brother, son have diabetes. Glyburide and pioglitazone were started, Insulin total daily dose was decreased from 90 units to 15 units a day. Her Hb A1c improved to 6 %, insulin was discontinued. She underwent Roux-en-Y Gastric Bypass surgery, lost 30 lbs. BMI improved to 42 kg/m2, Hb A1c became 5.3%. Her current glucose level is normal on Glyburide 2.5 mg and pioglitazone 7.5 mg daily. Results: Patient and his mother had heterozygous gene polymorphism in GLIS3, c.951G&amp;gt;T (p.Glu317Asp). This sequence replaces glutamic acid with aspartic acid at codon 317 of the GLIS3 protein (p.Glu317Asp). GLIS3 is a variant of MODY and is a transcriptional factor in beta cell differentiation and insulin production. Patient and mother had pathogenic heterozygous variant of KSR2 gene, c.1622del, resulting in a frameshift and premature protein termination (p.Lys541Serfs*27). Pathogenic variants in KSR2 are associated with obesity and insulin resistance. Patient was also heterozygous in the NTRK2 gene for a sequence variant c.500C&amp;gt;A, which is predicted to result in the amino acid substitution p.Ser167Tyr, also associated with obesity. Conclusion Genetic analysis of MODY is necessary even in cases of diabetes with morbid obesity, negative islet cell AB, positive family history of diabetes. Diagnosis of MODY helps provide individualized treatment and avoid complications secondary to unnecessary treatment with insulin. Presentation: Thursday, June 15, 2023

A Modified Differentiation Protocol In Vitro to Generate Dopaminergic Neurons from Pluripotent Stem Cells

Cell transplantation is considered a promising therapeutic strategy for the treatment of Parkinson's disease. Because of their strong differentiation potential, pluripotent stem cells may become a source of dopaminergic neurons for cell transplantation. Although published protocols have revealed that pluripotent stem cells can be successfully induced into dopaminergic neurons, unwanted cell types still exist in PSC-derived cultures. Therefore, signaling parameters for dopaminergic neuron patterning in differentiation protocols need to be further identified and optimized. In this study, we explored an In Vitro modified differentiation protocol for efficiently inducing dopaminergic neurons from pluripotent stem cells. Briefly, pluripotent stem cells were incubated in N2B27 medium for a 4-day culture, and then bFGF, SHH-C24II, purmorphamine, FGF8a and laminin were added to the medium. After a 6-day culture, the medium was replaced with N2B27 medium containing L-ascorbic acid, glial cell line-derived neurotrophic factor, cyclic adenosine monophosphate, laminin, and brain-derived neurotrophic factor for an additional 10 days. We confirmed that combined treatment with bFGF, SHH-C24II, purmorphamine, FGF8a and laminin significantly promoted the differentiation of pluripotent stem cells into dopaminergic neurons. Additionally, we determined a reasonable time window for the use of these factors. Our study provides new insights into the role of cell factors in dopaminergic neuron differentiation of pluripotent stem cells.

Engineering E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells.

Naturally occurring protein nanocages are promising drug carriers as both the interior and exterior can be decorated for drug encapsulation and cell targeting. To provide surface functionalization, we added a SpyTag to E2 nanocages (ST-E2) to enable tunable decoration using the robust SpyCatcher bioconjugation strategy. Additionally, the E2 core was mutated with four phenylalanine substitutions for doxorubicin loading and pH-responsive release. By decorating the exterior with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SpyCatcher, we demonstrated targeted cell death in inflammatory breast cancer cells compared to healthy breast epithelial cells at concentrations below the IC50 of free doxorubicin.

A signal R3-type, CAPRICE-like MYB transcription factor from Dendrobium nobile controls trichome and root-hair development in Arabidopsis

The CAPRICE-like MYB transcription factors with R3 MYB motif play a central role in regulating trichome and root-hair development in plants. We identified the homologous gene of ENHANCER OF TRY AND CPC (ETC) in Arabidopsis from Dendrobium nobile Lindl with full cDNA sequence and genomic sequence (CAPRICE-LIKE MYB, DnCPL and DngCPL) respectively. Phylogenic analyses revealed a close relationship of CAPRICE-like MYB TFs between D. nobile and A. thaliana. Promoter analysis indicated that DnCPL is specifically expressed in trichome basal cells of leaf epidermis and root hairs. Overexpression of DnCPL results in the suppression of trichome formation and overproduction of root hairs. In transgenic plants overexpressing DnCPL and DngCPL, trichome formation was inhibited, moreover, no trichomes were observed in tissues of aerial parts, and root-hair differentiation was significantly enhanced by strongly repressing endogenous genes of AtCPC, AtTCL1, and AtTCL2 expression, thereby enhancing AtTRY expression. The DnCPL RNAi plants formed fewer lateral roots with a corresponding change in AtCPC, AtTCL1 and AtTCL2 expression. These results suggest that Dendrobium and Arabidopsis partially use similar transcription factors for epidermal cell differentiation and the CPC-like R3 MYB, DnCPL, may be a key common regulator of plant trichome and root-hair development. The results also provided genes and means of regulation to improve the survival ratio of artificially cultivated Dendrobium with more lateral roots.

LSR targets YAP to modulate intestinal Paneth cell differentiation

Lipolysis-stimulated lipoprotein receptor (LSR) is a multi-functional protein that is best known for its roles in assembly of epithelial tricellular tight junctions and hepatic clearance of lipoproteins. Here, we investigated whether LSR contributes to intestinal epithelium homeostasis and pathogenesis of intestinal disease. By using multiple conditional deletion mouse models and exvivo cultured organoids, we find that LSR elimination in intestinal stem cells results in the disappearance of Paneth cells without affecting the differentiation of other cell lineages. Mechanistic studies reveal that LSR deficiency increases abundance of YAP by modulating its phosphorylation and proteasomal degradation. Using gain- and loss-of-function studies, we show that LSR protects against necrotizing enterocolitis through enhancement of Paneth cell differentiation in small-intestinal epithelium. Thus, this study identifies LSR as an upstream negative regulator of YAP activity, an essential factor for Paneth cell differentiation, and a potential therapeutic target for necrotizing enterocolitis.

Transplantable stem cell nanobridge scaffolds for accelerating articular cartilage regeneration

Microfracture technique for treating articular cartilage defects usually has poor clinical outcomes due to critical heterogeneity and extremely limited in quality. To improve the effects of current surgical technique (i.e., microfracture technique), we propose the transplantable stem cell nanobridge scaffold, acting as a protective bridge between host tissue and defected cartilage as well as microfracture-derived cells. Nanobridge scaffolds have a sophisticated nanoaligned structure with freestanding and flexible shapes for imposing direct structural guidance to cells including transplanted stem cells and host cells, and it can induce not only chondrocyte migration but also stem cell differentiation, maturation, and growth factor secretion. The transplantable stem cell nanobridge scaffold is capable of reconstructing the defected cartilage with homogeneous architecture and highly enhanced adhesive stress similar with native cartilage tissue by the synergistic effects of stem cells-based chondro-induction and nanotopography-based chondro-conduction. Our findings demonstrate a significant advancement in the traditional treatment technique by using a nanoengineered tool for achieving successful cartilage regeneration.

Neurodevelopmental risk and adaptation as a model for comorbidity among internalizing and externalizing disorders: genomics and cell-specific expression enriched morphometric study

BackgroundComorbidity is the rule rather than the exception for childhood and adolescent onset mental disorders, but we cannot predict its occurrence and do not know the neural mechanisms underlying comorbidity. We investigate if the effects of comorbid internalizing and externalizing disorders on anatomical differences represent a simple aggregate of the effects on each disorder and if these comorbidity-associated cortical surface differences relate to a distinct genetic underpinning.MethodsWe studied the cortical surface area (SA) and thickness (CT) of 11,878 preadolescents (9–10 years) from the Adolescent Brain and Cognitive Development Study. Linear mixed models were implemented in comparative and association analyses among internalizing (dysthymia, major depressive disorder, disruptive mood dysregulation disorder, agoraphobia, panic disorder, specific phobia, separation anxiety disorder, social anxiety disorder, generalized anxiety disorder, post-traumatic stress disorder), externalizing (attention-deficit/hyperactivity disorder, oppositional defiant disorder, conduct disorder) diagnostic groups, a group with comorbidity of the two and a healthy control group. Genome-wide association analysis (GWAS) and cell type specificity analysis were performed on 4468 unrelated European participants from this cohort.ResultsSmaller cortical surface area but higher thickness was noted across patient groups when compared to controls. Children with comorbid internalizing and externalizing disorders had more pronounced areal reduction than those without comorbidity, indicating an additive burden. In contrast, cortical thickness had a non-linear effect with comorbidity: the comorbid group had no significant CT differences, while those patient groups without comorbidity had significantly higher thickness compare to healthy controls. Distinct biological pathways were implicated in regional SA and CT differences. Specifically, CT differences were associated with immune-related processes implicating astrocytes and oligodendrocytes, while SA-related differences related mainly to inhibitory neurons.ConclusionThe emergence of comorbidity across distinct clusters of psychopathology is unlikely to be due to a simple additive neurobiological effect alone. Distinct developmental risk moderated by immune-related adaptation processes, with unique genetic and cell-specific factors, may contribute to underlying SA and CT differences. Children with the highest risk but lowest resilience, both captured in their developmental morphometry, may develop a comorbid illness pattern.

The role of TET2-mediated ROBO4 hypomethylation in the development of diabetic retinopathy

BackgroundIn diabetic retinopathy, increasing evidence points to a link between the pathogenesis of retinal microangiopathy and the endothelial cell-specific factor roundabout4 (ROBO4). According to earlier research, specificity protein 1 (SP1) enhances the binding to the ROBO4 promoter, increasing Robo4 expression and hastening the progression of diabetic retinopathy. To determine if this is related to aberrant epigenetic modifications of ROBO4, we examined the methylation level of the ROBO4 promoter and the corresponding regulatory mechanism during the course of diabetic retinopathy and explored the effect of this mechanism on retinal vascular leakage and neovascularization.MethodsThe methylation level of CpG sites in the ROBO4 promoter was detected in human retinal endothelial cells (HRECs) cultured under hyperglycemic conditions and retinas from streptozotocin-induced diabetic mice. The effects of hyperglycemia on DNA methyltransferase 1, Tet methylcytosine dioxygenase 2 (TET2), 5-methylcytosine, 5-hydroxymethylcytosine, and the binding of TET2 and SP1 to the ROBO4 promoter, as well as the expression of ROBO4, zonula occludens 1 (ZO-1) and occludin were examined. Short hairpin RNA was used to suppress the expression of TET2 or ROBO4 and the structural and functional changes in the retinal microvascular system were assessed.ResultsIn HRECs cultured under hyperglycemic conditions, the ROBO4 promoter methylation level decreased. Hyperglycemia-induced TET2 overexpression caused active demethylation of ROBO4 by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, which enhanced the binding of SP1 to ROBO4, increased the expression of ROBO4, and decreased the expression of ZO-1 and occludin, leading to the abnormalities in monolayer permeability, migratory ability and angiogenesis of HRECs. The above pathway was also demonstrated in the retinas of diabetic mice, which caused leakage from retinal capillaries and neovascularization. Inhibition of TET2 or ROBO4 expression significantly ameliorated the dysfunction of HRECs and retinal vascular abnormalities.ConclusionsIn diabetes, TET2 can regulate the expression of ROBO4 and its downstream proteins by mediating active demethylation of the ROBO4 promoter, which accelerates the development of retinal vasculopathy. These findings suggest that TET2-induced ROBO4 hypomethylation is a potential therapeutic target, and anti- TET2/ROBO4 therapy is anticipated to emerge as a novel strategy for early intervention and delayed progression of diabetic retinopathy.

GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression.

T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2 Cre and Tbx21 Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3-sufficient and Gata3-deficient CNS-infiltrating CD4+ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.

#3760 CYTOSKELETON-ORGANELLE INTERACTION IN LIVING CELL EXPRESSING INF2 VARIANTS CAUSING FSGS AND PERIPHERAL NEUROPATHY

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury and represents a leading cause of ESRD. Studies with genetic FSGS show that a majority of genes are associated with regulation of podocyte cytoskeletons. INF2 is an actin assembly factor that mediates both polymerization and depolymerization and is the most frequent cause of dominant inherited FSGS. INF2 mutations located in the proximal half of DID (residues Leu57 to Glu184) typically cause CMT with early-onset FSGS, whereas that situating in the distal half (residues Glu184 to Leu245) cause FSGS alone. Previous studies disclosed that INF2 mutations primarily disorganize actin network and alter mitochondria shapes and vesicle motility. However, it remains elusive how the cytoskeletal network is concerted with the organelle morphology and functions in the disease cells. This study aims to characterize the interaction between cytoskeleton and organelle in cells expressing INF2 FSGS or CMT/FSGS variants. Method We characterized the cytoskeletal-organelle interaction in live HeLa or COS-7 cells expressing pathogenic INF2 variants in our cohort of FSGS. We performed a triple labeling by co-transfection of GFP-INF2, cytoskeletal markers (Lifeact-mCherry or EMTB-mScarletI), and by the uptake of organelle markers (Lysotracker or Mitotracker). To clarify the detailed pattern and dynamics of cytoskeletons and organelles, we employed the Spinning Disk Confocal Microscope DragonFly, which allows real-time super-resolution imaging with 70nm resolution. Results Wild-type-INF2 (WT-INF2) cells showed an ER pattern with perinuclear, Golgi accumulation. Mild INF2 variants causing FSGS alone (T161N, N202S) preserved the subcellular distribution essential similar to WT-INF2, while showing focal irregularity of ER networks. In contrast, severe INF2 variants leading to CMT/FSGS (G73D) exhibited a diffuse coarse ER pattern with few Golgi clusters and occasional peripheral accumulation in both cell poles. In live imaging of cells coexpressing GFP-INF2 and Lifeact-mCherry, actin stress fibers were pronouncedly reduced in CMT/FSGS variant (G73D) cells, but were mildly decreased FSGS variant (T161N and N202S) cells, compared with WT-INF2 cells. The filopodia in cells expressing T161N variant were less mobile (n = 10, P&amp;lt; 0.01) but were longer (n = 10, P&amp;lt; 0.0001) than those in cells expressing WT-INF2. T161N variant reduced expression of cortactin that stabilizes actin-branching and narrowed area of cortical actin meshwork compared to the WT-INF2 (n = 11, P&amp;lt; 0.01), suggesting that INF2 could serve the cortical actin formation. HeLa cells coexpressing T161N variant and EMTB showed a bipolar cell elongation along the longitudinal axis (n≥30, P&amp;lt; 0.0001) and disarranged microtubule bundles aligned in parallel, compared with those expressing WT-INF2. A time-lapse imaging of living COS-7 cells revealed that T161N variant increases mitochondrial fragmentation as well as mitochondria-ER interfaces more prominent than WT-INF2. Moreover, T161N variant induced the mitochondria misdistribution at the cell periphery (filopodia and cell edge) in both cell poles. Such aberrant patterns frequently coexisted with disarranged microtubules. Moreover, in lysotracker-labeled living COS-7, T161N variant reduced the motility of the lysosomal vesicles in cortical actin meshwork area, compared with WT-INF2 (n = 100, P&amp;lt; 0.0001). These observations suggest that INF2 variants perturbate the trafficking of organelles through disorganization of actin-microtubule network. Conclusion Our results indicate that INF2 CMT/FSGS variants cause more global and severe organelle dysfunction by disrupting the organelle-cytoskeletal interaction, than INF2 FSGS variants. The microtubule disarrangement might be due to an insufficient capping mechanism by which the microtubules are stably tethered to the cortical actin meshwork. Elucidation of cell-specific factors will help better understanding why podocyte and Schwann cell are susceptible to the cytoskeletal disarrangement.

Novel perspectives on nanotechnological and biomedical implications of monotherapy or combination regimen of lactoferrin.

Lactoferrin (LF) is a protein molecule with a wide variety of physiological properties. LF has broad-spectrum antibacterial, antiviral, antioxidant, and antitumor, and possesses immunomodulatory properties to regulate immunity and gastrointestinal function. The main aim of this review is to explore the recent investigations on the functional role of LF against several human disorders and diseases through monotherapy or combinatorial regimens with other biological/chemotherapeutic agents through novel nanoformulations. We significantly searched public databases such as Pubmed, National Library of Medicine, relemed, Scopus and collected published reports pertaining to these recent reports on lactoferrin as a monotherapy or combination therapy, and its nanoformulations. We have discussed vividly the role of LF as a growth factor with substantial potential that can promote cell growth and regeneration potential for repairing tissues such as bone, skin, mucosa, and tendons. In addition, we have discussed novel perspectives on the role of LF as an inductive factor for the proliferation of stem cells in tissue recovery and discussed its novel modulating effects in ameliorating cancer and microbial growth through several signaling cascades via monotherapy or combinatorial regimens. Furthermore, the regeneration potential of this protein is reviewed to explore the efficacy and prospects of new treatment methods. This review benefits various microbiologists, stem cell therapists, and oncologists to explore the efficacy of LF in several segments of medicine by examining its ability as a stem cell differentiation factor, and anticancer agent or antimicrobial agent through novel formulations in preclinical or clinical study.

Japanese Laws and the Current Status of Regenerative Medicine in the Tohoku Region.

The aim of this study was to review Japanese laws regarding regenerative medicine and the current status of clinical application of regenerative medicine, to learn about the advantages and problems, and to thereby serve as a reference for measures necessary for the development of regenerative medicine. Regenerative medicine started in 1957 with the transplantation of hematopoietic stem cells, followed by the establishment of embryonic stem cells in 1981 and induced pluripotent stem cells in 2006, and continues to evolve progressively. At the same time, however, problems have emerged due to lax legal regulations, such as the use of treatments that lack scientific evidence. The Japanese government enacted two laws to regulate regenerative medicine: the Law to Ensure the Safety of Regenerative Medicine and the Amend the Pharmaceutical Affairs Law in 2013. These laws were enacted with the aim of providing safe regenerative medicine promptly and smoothly and developing many regenerative medicine products. In these laws, regenerative medicine is defined as medical treatment that restores lost functions of damaged organs and tissues with the help of cellular and tissue-based products. Nowadays, there are two major methods of regenerative medicine. One representative method involves the transplantation of devices that activates self-regenerative ability by introducing living cells into patients' body. The other method is the activation and differentiation of endogenous stem cells with cell growth and differentiation factors. The current status of regenerative medicine in the Tohoku region after the enactment of these laws is described in detail. This clarified the advantages and disadvantages associated with regenerative medicine as it is currently practiced in Japan. Development of regenerative medicine in dentistry will be advanced by learning about its clinical application in medicine.

Fabrication of ECM Mimicking Bioactive Scaffold: A Regenerative Approach for MSC Mediated Applications.

Biomaterials are feasible resources that aids to replace damaged structures in our bodies. The most biologically active flora is Aloe vera which has many bioactive compounds that are anti-inflammatory, antimicrobial, and have ECM mimicking protein content which helps in the healing of wounds and also acts as an ECM factor for stem cell homing and differentiation. The Aloe vera containing 10 w/v of gelatin was lyophilized. Scaffolds had sharper morphology, greater hydrophilic properties, and a Young's modulus of 6.28 MPa and 15.9 MPa of higher tensile strength are desirable. In tissue engineering and regenerative medicine, biologically active scaffolds have been producing hopeful outcomes in both restoration and replacement, respectively. The objective of the present investigation is to test the idea that incorporating gelatin to Aloe vera scaffolds might enhance their structure, good biocompatibility, and possibly even bioactivity. The SEM picture of the composite scaffold revealed pore walls. The scaffolds had linked pores with diameters ranging from 93 to 296 μm. Aloe vera and the matrix interact well, according to the FTIR study, which could lead to a reduction in the amount of water-binding sites and a reduction in the material's ability to absorb water. Aloe vera with 10% gelatin (AV/G) scaffold was investigated for different biological reactions of human gingival tissue mesenchymal stem cells (MSCs) in terms of cell proliferation, morphology, and cell migration. The results demonstrated the potential of the AV/G scaffold as a biomaterial that offers new insight in the field of tissue engineering.

An essential role of iron in Tfh cell development and effector function

Abstract Background: T follicular helper CD4+ cells (Tfh) are essential for maintenance of systemic lupus erythematosus (SLE) as they help germinal center B cells generate high affinity auto-antibodies. The activation of STAT3 is critical for generation of Tfh cells as it promotes the transcription of BCL6 (the essential transcription factor for Tfh cell differentiation). It is recognized that activated, proliferating SLE CD4+ T cells, contain more iron than controls. Iron binds CDK1 to accentuate JAK1 phosphorylation and activation of STAT3 in proliferating cells. Whether increased iron content of SLE CD4+ T cells augments JAK1-STAT3 signaling and their differentiation into Tfh cells has not been investigated. Experiments: RNAseq and qPCR was used to compare iron regulatory pathways in purified Tfh cells from WT and SLE-prone mice. Labile iron (bio-active) content was measured in freshly isolated Tfh cells. The effect of iron chelator on in vitro Tfh differentiation and effector function was evaluated. Results: Compared to WT, SLE Tfh cells displayed significant increase in genes associated with iron uptake and transmembrane iron transport. Labile iron content and CDK1 gene expression were elevated in SLE Tfh cells. Iron chelation attenuated STAT3 activation, BCL6 expression and IL-21 production in in vitro generated Tfh cells. Conclusion: Our novel data identify an essential role of iron in Tfh cell differentiation and effector function. Increased iron and CDK1 may amplify JAK1-STAT3 signaling and potentiate Tfh cell differentiation. This can be blocked by restricting free iron availability. Understanding this aspect of Tfh cell biology in SLE may support strategies that control labile iron as potential adjunct therapy. Supported by grants from Vifor Pharma (P0213104, P0226952) to Yogesh Scindia NIH (R01AI135128) to Borna Mehrad NIH (R01AI128901) to Laurence Morel