Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor.

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.

Glucosamine and Silibinin Alter Cartilage Homeostasis through Glycosylation and Cellular Stresses in Human Chondrocyte Cells.

Osteoarthritis is more prevalent than any other form of arthritis and is characterized by the progressive mechanical deterioration of joints. Glucosamine, an amino monosaccharide, has been used for over fifty years as a dietary supplement to alleviate osteoarthritis-related discomfort. Silibinin, extracted from milk thistle, modifies the degree of glycosylation of target proteins, making it an essential component in the treatment of various diseases. In this study, we aimed to investigate the functional roles of glucosamine and silibinin in cartilage homeostasis using the TC28a2 cell line. Western blots showed that glucosamine suppressed the N-glycosylation of the gp130, EGFR, and N-cadherin proteins. Furthermore, both glucosamine and silibinin differentially decreased and increased target proteins such as gp130, Snail, and KLF4 in TC28a2 cells. We observed that both compounds dose-dependently induced the proliferation of TC28a2 cells. Our MitoSOX and DCFH-DA dye data showed that 1 µM glucosamine suppressed mitochondrial reactive oxygen species (ROS) generation and induced cytosol ROS generation, whereas silibinin induced both mitochondrial and cytosol ROS generation in TC28a2 cells. Our JC-1 data showed that glucosamine increased red aggregates, resulting in an increase in the red/green fluorescence intensity ratio, while all the tested silibinin concentrations increased the green monomers, resulting in decreases in the red/green ratio. We observed increasing subG1 and S populations and decreasing G1 and G2/M populations with increasing amounts of glucosamine, while increasing amounts of silibinin led to increases in subG1, S, and G2/M populations and decreases in G1 populations in TC28a2 cells. MTT data showed that both glucosamine and silibinin induced cytotoxicity in TC28a2 cells in a dose-dependent manner. Regarding endoplasmic reticulum stress, both compounds induced the expression of CHOP and increased the level of p-eIF2α/eIF2α. With respect to O-GlcNAcylation status, glucosamine and silibinin both reduced the levels of O-GlcNAc transferase and hypoxia-inducible factor 1 alpha. Furthermore, we examined proteins and mRNAs related to these processes. In summary, our findings demonstrated that these compounds differentially modulated cellular proliferation, mitochondrial and cytosol ROS generation, the mitochondrial membrane potential, the cell cycle profile, and autophagy. Therefore, we conclude that glucosamine and silibinin not only mediate glycosylation modifications but also regulate cellular processes in human chondrocytes.

Basal differentiation and expression status of SOX2 and KLF4 in basal layers are prognostic factors for disease-specific survival in oral squamous cell carcinoma.

BACKGROUND: Basal differentiation in oral squamous cell carcinoma is usually detected at invasive sites. However, its significance as a prognostic value has been poorly investigated. METHODS: COL17 was selected as a basal differentiation marker because of its stable expression in the basal-like cells of oral squamous cell carcinoma. Sixty-five cases of oral squamous cell carcinoma were subclassified into COL17-high (30 cases) and -low (35 cases) types, and the prognostic value was analyzed by Cox regression analysis. In addition, the stem cell markers such as SOX2, KLF4, MYC as well as the stem cell-related markers BMI1, EZH2, and YAP and its paralog TAZ, were immunohistochemically analyzed. Their prognostic values were investigated along with their COL17 status by Cox regression analysis. RESULTS: No significant difference was observed between the COL17-high and -low groups in the disease-specific survival and recurrence-free survival in oral squamous cell carcinoma. When the COL17-high and -low categories were combined with the SOX2, KLF4, EZH2, or YAP/TAZ status in the basal layers, together with gender and age as covariates, the hazard ratios reached 3.3, 3.7, 2.8, and 3.1, respectively. In addition, multivariate analysis, including COL17, SOX2, and KLF4, with gender and age as covariates, showed a significantly poor prognosis for disease-specific survival. CONCLUSION: Based on the relatively high hazard ratios, it is indicated that basal differentiation and the expression status of SOX2 and KLF4 in the basal layers are prognostic factors for oral squamous cell carcinoma.

Meningioma: current updates on genetics, classification, and mouse modeling.

Meningiomas, the most common primary brain tumors in adults, are often benign and curable by surgical resection. However, a subset is of higher grade, shows aggressive growth behavior as well as brain invasion, and often recurs even after several rounds of surgery. Increasing evidence suggests that tumor classification and grading primarily based on histopathology do not always accurately predict tumor aggressiveness and recurrence behavior. The underlying biology of aggressive treatment-resistant meningiomas and the impact of specific genetic aberrations present in these high-grade tumors is still only insufficiently understood. Therefore, an in-depth research into the biology of this tumor type is warranted. More recent studies based on large-scale molecular data such as whole exome/genome sequencing, DNA methylation sequencing, and RNA sequencing have provided new insights into the biology of meningiomas and have revealed new risk factors and prognostic subtypes. The most common genetic aberration in meningiomas is functional loss of NF2 and occurs in both low- and high-grade meningiomas, whereas NF2-wildtype meningiomas are enriched for recurrent mutations in TRAF7, KLF4, AKT1, PI3KCA, and SMO and are more frequently benign. Most meningioma mouse models are based on patient-derived xenografts and only recently have new genetically engineered mouse models of meningioma been developed that will aid in the systematic evaluation of specific mutations found in meningioma and their impact on tumor behavior. In this article, we review recent advances in the understanding of meningioma biology and classification and highlight the most common genetic mutations, as well as discuss new genetically engineered mouse models of meningioma.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Role of ONECUT family transcription factors in cancer and other diseases.

Members of ONECUT transcription factor play an essential role in several developmental processes, however, the atypical expression of ONECUT proteins lead to numerous diseases, including cancer. ONECUT family proteins promote cell proliferation, progression, invasion, metastasis, angiogenesis, and stemness. This family of proteins interacts with other proteins such as KLF4, TGF-ß, VEGFA, PRC2, SMAD3 and alters their expression involved in the regulation of various signaling pathways including Jak/Stat3, Akt/Erk, TGF-ß, Smad2/3, and HIF-1α. Furthermore, ONECUT proteins are proposed as predictive biomarkers for pancreatic and gastric cancers. The present review summarizes the involvement of ONECUT family proteins in the development and progression of various human cancers and other diseases.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Establishment of a transgene-free iPS cell line (SDCHi003-A) from a young patient bearing a NPRL2 mutation and suffering from Epilepsy.

Epilepsy affects âˆ¼ 65 million people worldwide. Status epilepticus can lead to life-threatening if untreated. In this study, peripheral blood mononuclear cells were isolated from a young patient patient bearing a Nitrogen Perntease Regulator Like 2 Protein (NPRL2) mutation and suffering from Epilepsy verified by clinical and genetic diagnosis. Induced pluripotent stem cells (iPSCs) were established by a non-integrative method, using plasmids carrying OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSCs presented typical pluripotent cells morphology, normal karyotype, and potential to differentiate into three germ layers. Our approach offers a useful model to explore pathogenesis and therapy of Epilepsy.

Generation of an induced pluripotent stem cell line GWCMCi006-A from a patient with autosomal dominant neurodevelopmental disorder with or without hyperkinetic movements and seizures harboring GRIN1 c.389A > G mutation.

Autosomal dominant neurodevelopmental disorder with or without hyperkinetic movements and seizures (NDHMSD) is a rare neurological disorder characterized by neurodevelopmental disorder and hyperkinetic movement, with or without seizures. Heterozygous mutation in the GRIN1 encoding the subunit 1 of the N-methyl-D-aspartate receptor caused this disorder. We first established an induced pluripotent stem cell (iPSC) line from a male patient with c.389A > G mutation in the GRIN1, via reprogramming with KLF4, SOX2, OCT3/4, and c-MYC. Through identification examination, the iPSCs (GWCMCi006-A) stably expressed pluripotency-associated stem cell markers, maintained a normal karyotype, and showed proliferative potential for three-germ layers differentiation.

MircoRNA-25-3p in skin precursor cell-induced Schwann cell-derived extracellular vesicles promotes axon regeneration by targeting Tgif1.

Nerve injury often leads to severe dysfunction because of the lack of axon regeneration in adult mammal. Intriguingly a series of extracellular vesicles (EVs) have the obvious ability to accelerate the nerve repair. However, the detailed molecular mechanisms to describe that EVs switch neuron from a transmitter to a regenerative state have not been elucidated. This study elucidated the microRNA (miRNA) expression profiles of two types of EVs that promote nerve regeneration. The functions of these miRNAs were screened in vitro. Among the 12 overlapping miRNAs, miR-25-3p was selected for further analysis as it markedly promoted axon regeneration both in vivo and in vitro. Furthermore, knockdown experiments confirmed that PTEN and Klf4, which are the major inhibitors of axon regeneration, were the direct targets of miR-25-3p in dorsal root ganglion (DRG) neurons. The utilization of luciferase reporter assays and functional tests provided evidence that miR-25-3p enhances axon regeneration by targeting Tgif1. Additionally, miR-25-3p upregulated the phosphorylation of Erk. Furthermore, Rapamycin modulated the expression of miR-25-3p in DRG neurons. Finally, the pro-axon regeneration effects of EVs were confirmed by overexpressing miR-25-3p and Tgif1 knockdown in the optic nerve crush model. Thus, the enrichment of miR-25-3p in EVs suggests that it regulates axon regeneration, proving a potential cell-free treatment strategy for nerve injury.

miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating cancer stem cells.

Cancer stem cells (CSCs) are known for their potent ability to drive tumor initiation and recurrence, yet the molecular mechanisms regulating CSCs are still unclear. Our study found a positive correlation between increased levels of miR-29a and better survival rates in early-stage breast cancer patients, but a negative correlation in late-stage patients, suggesting a dual function of miR-29a in regulating breast cancer. Furthermore, miR-29a showed significant downregulation in the ALDH+ breast cancer stem cell population compared to non-stem cancer cells. Overexpression of miR-29a in human breast cancer cells reduced the proportion of CSCs, suppressed their ability to form mammospheres, and inhibited the expression of stemness genes SOX2, KLF4, and hTERT in vitro. Conversely, knockdown of miR-29a in breast cancer cells showed opposite effects. Tumor xenograft experiments revealed that miR-29a overexpression significantly inhibited tumorigenesis initiated by MDA-MB-231 cell transplantation in nude mice. We further demonstrated that Krüppel-like factor 4 (KLF4), a key gene that regulates cell stemness, was a direct target of miR-29a in breast cancer cells. miR-29a suppressed the expression of KLF4 at both mRNA and protein levels. Reintroduction of KLF4 into breast cancer cells rescued the miR-29a-induced CSC suppression phenotype. In summary, our study is the first to demonstrate that miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating CSCs, which provides novel therapeutic targets for preventing breast tumor initiation.

Zishen Qingre Lishi Huayu recipe promotes proliferation and inhibits apoptosis of GCs of PCOS via KLF4-C/EBPß pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zishen Qingre Lishi Huayu recipe (ZQLHR) is a herbal recipe created on the basis on the theory of traditional Chinese medicine and clinical practice, and is mainly used in the treatment of polycystic ovary syndrome (PCOS). However, the underlying mechanism for this fact has not been clearly elucidated. AIM OF THE STUDY: To verify whether ZQLHR regulates granulosa cells (GCs) proliferation and apoptosis through the Krüppel-like factor 4 (KLF4) - CCATT enhancer-binding proteinß (C/EBPß) pathway, and to provide in vitro molecular mechanism supporting for the effects of ZQLHR to enhance follicular development and treat patients with PCOS. MATERIALS AND METHODS: Based on previous experiments, we performed the following experiments. Firstly, we treated KGN cells (a steroidogenic human granulosa-like tumor cell line) for 48 h using different concentrations of ZQLHR in order to observe apoptosis in each group. Secondly, the mRNA and protein expression levels of KLF4 and C/EBPß in KGN cells after administrated with ZQLHR were examined by quantitative real-time PCR(q-PCR) and Western blot assay. Thirdly, after knocking down KLF4 and C/EBPß using siRNAs, the relationship between KLF4 and C/EBPß in KGN cells was detected. Further, cell counting kit-8 assay, colony formation assay and flow cytometry were used to verify whether ZQLHR promotes proliferation and facilitates apoptosis in KGN cells through the KLF4-C/EBPß pathway. Finally, q-PCR and Western blot were used to test whether ZQLHR mediated proliferation and apoptosis-related factors such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (BAX), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 to affect the proliferation and apoptosis of KGN cells through the KLF4-C/EBPß pathway. CONCLUSIONS: ZQLHR, containing 0.2% by volume, administered to KGN cells resulted in the lowest rate of apoptosis. The expression levels of KLF4 and C/EBPß were increased in KGN cells following ZQLHR treatment. Additionally, ZQLHR promoted proliferation and inhibited apoptosis of KGN cells by modulating proliferation and apoptosis-related factors via the KLF4-C/EBPß pathway. Furthermore, we confirmed that KLF4 and C/EBPß regulate each other in KGN cells. These findings indicate that ZQLHR enhances the proliferation of GCs and suppresses their apoptosis, which constitutes a therapeutic mechanism for treating patients with PCOS.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Generation of induced pluripotent stem cell line NIMHi010-A from dermal fibroblast cells of a healthy individual.

In this study, we have established human induced pluripotent stem cell (hiPSC) line, NIMHi010-A of a 42-year-old healthy donor. The iPSC line was generated from human dermal fibroblasts using Sendai viruses carrying reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The generated hiPSC line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line will serve as a healthy control model for physiological processes and drug screening of Asian origin from Indian population.

Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells.

Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Generation of induced pluripotent stem cell line (VRISGi004-A) from a healthy female donor by reprogramming erythroid progenitor cells.

We generated a human induced pluripotent stem cell (hiPSC) line from erythroid progenitor cells (EPCs) of a 20-year-old female healthy donor using Sendai virus vector encoding Yamanaka factors OCT3/4, SOX2, c-MYC, and KLF4. The established hiPSCs showed a standard morphology and expression of typical undifferentiated stem cell markers, a normal karyotype (46, XX), and demonstrated potential for differentiation in vitro. Furthermore, they were successfully differentiated into cardiomyocytes that expressed cardiomyocyte-specific markers. The iPSC line and iPSC-derived cardiomyocytes will provide new avenues for future drug testing/development and personalized cell therapy for cardiovascular diseases (CVDs).

Exogenous PRAS40 reduces KLF4 expression and alleviates hypertrophic scar fibrosis and collagen deposition through inhibiting mTORC1.

BACKGROUND: To identify the anti-fibrosis effect of PRAS40 in scar, and its potential mechanism. METHODS: We constructed a rat model of hypertrophic scarthat was locally injected the PRAS40 overexpression adenoviruses, mTORC1 inhibitor MHY1485 and activator rapamycin, and further observed the pathological changes of skin tissue and the severity of fibrosis by HE, Masson and sirius red staining, and analyzed the deposition of a-SMA and collagen I by western blot and immunofluorescence test. Meanwhile, the co-localization of KLF4 with a-SMA and type I collagen was analyzed, as well as the regulatory effect of PRAS40 on KLF4. In addition, we also verified whether the inhibition of scar fibrosis by PRAS40 is related to mTORC1, and whether the upregulation of KLF4 is related to mTORC1. RESULTS: The results showed that the expression of PRAS40 was low and p-PRAS40 was high in scar skin tissue. After local injection of PRAS40 overexpression adenovirus, the expression of PRAS40 in skin tissue was increased. The overexpression of PRAS40 can inhibit scar skin fibrosis and reduce the content of a-SMA and collagen I. Further mechanism analysis confirms that the inhibitory effect of PRAS40 on skin fibrosis is related to mTORC1, and PRAS40 inhibits the activation of mTORC1. The expression of KLF4 is relatively low in scar tissue. PRAS40 administration upregulated the expression of KLF4, which is related to mTORC1 CONCLUSIONS: PRAS40 significantly improves fibrosis of scar skin tissue and increases the expression of KLF4 in scars. The anti-fibrotic effect of PRAS40 depends on mTORC1.

Generation of two hiPSCs lines of two patients carrying truncating mutations in the dimerization domain of filamin C.

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi004-A and HIMRi005-A from dermal fibroblasts of a 48-year-old female (HIMRi004-A) carrying missense mutation that translate to the first described filamin C isoform p.W2710X and from a 56-year-old female (HIMRi005-A) carrying a recently described mutation in the same domain p.Y2704X. Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. The lines display a typical embryonic stem cell-like morphology, express pluripotency markers, retain a normal karyotype (46, XX) and have the differentiation capacity in all three germ layers. The two lines can be used to elucidate the pathomechanisms of FLNC myofibrillar myopathies and to develop novel therapeutic options.

KLF4 interacts with TXNIP to modulate the pyroptosis in ulcerative colitis via regulating NLRP3 signaling.

INTRODUCTION: Ulcerative colitis (UC) is one of the most common diseases in the gastrointestinal tract related to abnormal inflammation. Pyroptosis, which is characterized by the formation of inflammasome, activation of caspase-1, and separation of N- and C-terminus of gasdermin D (GSDMD), and may be involved in the pathogenesis of IBD. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in differentiated epithelial cells. KLF4 mediates proinflammatory signaling in macrophages. Here, we tested whether KLF4 is functional in pyroptosis of UC. METHODS: In human UC tissues and/or lipopolysaccharide (LPS)/adenosine 5-triphosphate (ATP) stimulation human colon epithelial cells, KLF4, TXNIP, Cleave-Caspase-1, and GSDMD expression were detected through quantitative reverse transcription polymerase chain reaction (PCR), immunohistochemical and western blot assay. Interleukin (IL)-1ß and IL-18 levels were quantified by enzyme-linked immunosorbent assay. We successfully constructed a KLF4-silenced colon epithelial cell line using an adenovirus vector. We apply the UCSC and JASPAR to predict the KLF4 binding sites in the promoter region of TXNIP. RESULTS: In human UC tissues and/or LPS/ATP stimulation human colon epithelial cells, KLF4, TXNIP, Caspase-1, and GSDMD expression level were significantly elevated via quantitative reverse transcription PCR, immunohistochemical and western blot assay. Moreover, We identified that there is an interaction between KLF4 and TXNIP through Yeast double hybrid assay and CO-IP assay. We successfully constructed a KLF4-silenced human intestinal epithelial cell line. In LPS/ATP stimulation KLF4-silenced human intestinal epithelial cells, KLF4, TXNIP, Cleave Caspase-1, ASC, and GSDMD expression level were significantly decreased via quantitative reverse transcription PCR. CONCLUSION: Our results confirm that KLF4 can positively regulate the expression of TXNIP and regulate the pyroptosis process of UC through the TXNIP/NLRP3 pathway.