Reproductive and transgenerational toxicity of bisphenol S exposure in pregnant rats: Insights into hormonal imbalance and steroid biosynthesis pathway disruption.

Bisphenol S (BPS) is an alternative chemical to bisphenol A commonly used in food packaging materials. It raises concerns due to potential adverse effects on human health. However, limited evidence exists regarding reproductive toxicity from BPS exposure, and the mechanism of associated transgenerational toxicity remains unclear. In this study, pregnant SD rats were exposed to two different doses of BPS (0.05 or 20 mg/kg) from GD6 to PND21. The objective was to investigate reproductive and transmissible toxicity induced by BPS, explore endocrine effects, and uncover potential underlying mechanisms in rats. Perinatal exposure to BPS in the F0 generation significantly decreased the rate of body weight, ovarian organ coefficient, and growth and development of the F1 generation. Notably, these changes included abnormal increases in body weight and length, estrous cycle disruption, and embryonic dysplasia in F1. 4D-DIA proteomic and PRM analyses revealed that exposure to 20 mg/kg group significantly altered the expression of proteins, such as Lhcgr and Akr1c3, within the steroid biosynthetic pathway. This led to elevated levels of FSH and LH in the blood. The hypothalamic-pituitary-ovarian (HPO) axis, responsible for promoting fertility through the cyclic secretion of gonadotropins and steroid hormones, was affected. RT-qPCR and Western blot results demonstrated that the expression of GnRH in the hypothalamus was decreased, the GnRHR in the pituitary gland was decreased, and the expression of FSHß and LHß in the pituitary gland was increased. Overall, BPS exposure disrupts the HPO axis, hormone levels, and steroid biosynthesis in the ovaries, affecting offspring development and fertility. This study provides new insights into the potential effects of BPS exposure on the reproductive function of the body and its relevant mechanisms of action.

TRAF3 deficiency in MDCK cells improved sensitivity to the influenza A virus.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein, has significant and varying effects on immunity depending on cell types. The role of TRAF3 in Madin-Darby Canine Kidney Epithelial (MDCK) cell resistance to influenza A virus (IVA) remains elusive. In the present study, CRISPR-Cas9 gene editing technology was used to construct the TRAF3 knockout MDCK cells (MDCK-TRAF3-/-). Hemagglutination assay, plaque assay, transcriptome, and quantitative real-time PCR were performed after IVA infection. The results showed that after IVA infection, HA titers and virus titers were promoted, interferon I-related pathways were significantly blocked, and transcription of several antiviral-related genes was significantly decreased in MDCK-TRAF3-/- cells. Thus, our study suggests that TRAF3 gene knockout reduced MDCK cell's resistance to IVA, thereby resulting in a promising way for IVA isolation and vaccine manufacturing.

Online education satisfaction assessment based on cloud model and fuzzy TOPSIS.

During the COVID-19, colleges organized online education on a massive scale. To make better use of online education in the post-epidemic era, this paper conducts an online education satisfaction survey with four types of colleges and 129,325 students propose a fuzzy TOPSIS (technique for order preference by similarity to ideal solution) method based on the cloud model to rank the satisfaction of different colleges. Firstly, based on the characteristics of online education during the COVID-19, we build an evaluation indicator system from four dimensions: technology, instructor, learner and environment including, 10 indicators and 94 sub-indicators. Secondly, the cloud model is used to quantitatively describe the natural language and uncertainty in a large amount of assessment information. The cloud model generator is used for sub-indicators and achieves an effective and flexible conversion between linguistic information and quantitative values. The cloud model of indicators are presented by integrating the corresponding sub-indicators. The weights of indicators are determined by the entropy method based on the cloud model and possibility degree matrix, which eliminates the judgment of decision-makers and has great power for handling practical problems with unknown weight information. Finally, a fuzzy TOPSIS method based on the cloud model is proposed to rank the satisfaction of online education of different colleges. The proposed method is compared with other existing methods to shown its merits. The experimental result is consistent with the proportion of students who accept online education in the post-epidemic era. According to the second questionnaire, as the qualitative evaluation of the cloud model of indicators increases, the qualitative evaluation of satisfaction of different types of colleges will also increase. It indicates that the method proposed in this paper is practical.

Current knowledge of targeted-release budesonide in immunoglobulin A nephropathy: A comprehensive review.

Immunoglobulin A (IgA) nephropathy is a common autoimmune kidney disease. Accumulating studies showed that IgA nephropathy may be partially correlated with mucosal immune system dysfunction. Systemic corticosteroid treatment exerts an essential protective effect against renal deterioration in IgA nephropathy. However, long-term use of corticosteroids may cause systemic side effects. The novel targeted-release formulation (TRF) of budesonide has been shown to deliver the drug to the distal ileum with the aim of minimizing adverse events for patients with IgA nephropathy. In this review, we have summarized all the current evidence of the effects of TRF-budesonide protecting against IgA nephropathy. Three randomized controlled trials (RCTs), one cohort, two case reports, and an ongoing Phase 3 trial (Part B, NCT03643965), were under comprehensive review. These included studies demonstrated that TRF-budesonide could remarkably reduce proteinuria, hematuria, and creatinine, as well as preserve renal function. The local immunosuppressive effects exhibited by TRF-budesonide may represent a novel and promising approach to treating IgA nephropathy. However, the current evidence was only derived from limited trials. Therefore, more well-designed RCTs are still warranted to validate the curable profile of TRF-budesonide in treating IgA nephropathy.

Stimulus-Responsive Microfluidic Interface Enables Efficient Enrichment and Cytogenetic Profiling of Circulating Myeloma Cells.

Minimal residual disease (MRD) provides an independent prognostic factor for multiple myeloma (MM) patients. However, clinical MRD assays suffer from highly invasive sampling, insufficient detection sensitivity, and high cost. Herein, a stiMulus-Responsive ligand-Decorated microfluidic chip (MRD-Chip) was developed for efficient capture and controlled release of circulating myeloma cells (CMCs) in the peripheral blood for noninvasive myeloma evaluation. The CD138 antibody-decorated herringbone chip with a disulfide linker was designed to enhance the collision probability between blood cells and capture antibodies, leading to high capture efficiency of CMCs. More importantly, the captured CMCs can be nondestructively released via a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence in situ hybridization assay, we successfully identified the cytogenetic abnormalities (chromosome 1q21 amplification and p53 deletion) of CMCs in clinical samples. Overall, with the merits of noninvasive sampling, high capture efficiency (70.93%), high throughput (1.5 mL/h), and nondestructive release of target cells (over 90% viability) for downstream analysis, our strategy provides new opportunities for myeloma evaluation, such as prognosis assessment, efficacy monitoring, and mechanism research of disease relapse and drug resistance.

Rbm24 modulates adult skeletal muscle regeneration via regulation of alternative splicing.

Rationale: The adult skeletal muscle can self-repair efficiently following mechanical or pathological damage due to its remarkable regenerative capacity. However, regulatory mechanisms underlying muscle regeneration are complicated and have not been fully elucidated. Alternative splicing (AS) is a major mechanism responsible for post-transcriptional regulation. Many aberrant AS events have been identified in patients with muscular dystrophy which is accompanied by abnormal muscle regeneration. However, little is known about the correlation between AS and muscle regeneration. It has been reported that RNA binding motif protein 24 (Rbm24), a tissue-specific splicing factor, is involved in embryo myogenesis while the role of Rbm24 in adult myogenesis (also called muscle regeneration) is poorly understood. Methods: To investigate the role of Rbm24 in adult skeletal muscle, we generated Rbm24 conditional knockout mice and satellite cell-specific knockout mice. Furthermore, a cardiotoxin (CTX)-induced injury model was utilized to assess the effects of Rbm24 on skeletal muscle regeneration. Genome-wide RNA-Seq was performed to identify the changes in AS following loss of Rbm24. Results: Rbm24 knockout mice displayed abnormal regeneration 4 months after tamoxifen treatment. Using RNA-Seq, we found that Rbm24 regulated a complex network of AS events involved in multiple biological processes, including myogenesis, muscle regeneration and muscle hypertrophy. Moreover, using a CTX-induced injury model, we showed that loss of Rbm24 in skeletal muscle resulted in myogenic fusion and differentiation defects and significantly delayed muscle regeneration. Furthermore, satellite cell-specific Rbm24 knockout mice recapitulated the defects in regeneration seen in the global Rbm24 knockout mice. Importantly, we demonstrated that Rbm24 regulated AS of Mef2d, Naca, Rock2 and Lrrfip1 which are essential for myogenic differentiation and muscle regeneration. Conclusions: The present study demonstrated that Rbm24 regulates dynamic changes in AS and is essential for adult skeletal muscle regeneration.

[Determination of ten cephalosporins in bee products by solid phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry].

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantitative determination of ten cephalosporins in bee products, including honey, royal jelly, and lyophilized royal jelly powder, has been established. The samples were extracted with an acetonitrile-water (80:20, v/v) solution. After purification by solid phase extraction using an Oasis PRIME HLB cartridge, the extract was blown to dryness under a stream of nitrogen gas and then re-dissolved in 1 mL 0.1% (v/v) formic acid solution and methanol (95:5, v/v). The samples were separated on an Acquity UPLC BEH C18 column, using a mixture of 0.1% (v/v) formic acid solution and methanol as the mobile phase under gradient elution. The analysis was carried out using a positive electrospray ion source in the multiple reaction monitoring mode. The matrix-matched external standard method was applied to quantitative analysis. Good linear relationships were obtained for the ten cephalosporins in certain concentration ranges, and the correlation coefficients were more than 0.999. The limits of detection and limits of quantification for the ten cephalosporins were in the ranges 0.15-1.5 µg/kg and 0.5-5 µg/kg, respectively. The recoveries for the analytes in the bee products were in the range of 75.0%to 89.8%, with relative standard deviations of 1.4% to 4.6%. This method is characterized by a short analysis time and is suitable for the determination of cephalosporins in different bee products by virtue of its simplicity and reliability.

RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy.

RNA splicing contributes to a broad spectrum of post-transcriptional gene regulation during normal development, as well as pathological manifestation of heart diseases. However, the functional role and regulation of splicing in heart failure remain poorly understood. RNA binding protein (RBP), a major component of the splicing machinery, is a critical factor in this process. RNA binding motif protein 24 (RBM24) is a tissue-specific RBP which is highly expressed in human and mouse heart. Previous studies demonstrated the functional role of RBM24 in the embryonic heart development. However, the role of RBM24 in postnatal heart development and heart disease has not been investigated. In this paper, using conditional RBM24 knockout mice, we demonstrated that ablation of RBM24 in postnatal heart led to rapidly progressive dilated cardiomyopathy (DCM), heart failure, and postnatal lethality. Global splicing profiling revealed that RBM24 regulated a network of genes related to cardiac function and diseases. Knockout of RBM24 resulted in misregulation of these splicing transitions which contributed to the subsequent development of cardiomyopathy. Notably, our analysis identified RBM24 as a splice factor that determined the splicing switch of a subset of genes in the sacomeric Z-disc complex, including Titin, the major disease gene of DCM and heart failure. Together, this study identifies regulation of RNA splicing by RBM24 as a potent player in remodeling of heart during postnatal development, and provides novel mechanistic insights to the pathogenesis of DCM.

Quantitative Proteomics Study Reveals Changes in the Molecular Landscape of Human Embryonic Stem Cells with Impaired Stem Cell Differentiation upon Exposure to Titanium Dioxide Nanoparticles.

The increasing number of nanoparticles (NPs) being used in various industries has led to growing concerns of potential hazards that NP exposure can incur on human health. However, its global effects on humans and the underlying mechanisms are not systemically studied. Human embryonic stem cells (hESCs), with the ability to differentiate to any cell types, provide a unique system to assess cellular, developmental, and functional toxicity in vitro within a single system highly relevant to human physiology. Here, the quantitative proteomics approach is adopted to evaluate the molecular consequences of titanium dioxide NPs (TiO2 NPs) exposure in hESCs. The study identifies ≈328 unique proteins significantly affected by TiO2 NPs exposure. Proteomics analysis highlights that TiO2 NPs can induce DNA damage, elevated oxidative stress, apoptotic responses, and cellular differentiation. Furthermore, in vivo analysis demonstrates remarkable reduction in the ability of hESCs in teratoma formation after TiO2 NPs exposure, suggesting impaired pluripotency. Subsequently, it is found that TiO2 NPs can disrupt hESC mesoderm differentiation into cardiomyocytes. The study unveils comprehensive changes in the molecular landscape of hESCs by TiO2 NPs and identifies the impact which TiO2 NPs can have on the pluripotency and differentiation properties of human stem cells.

Genome-wide trait-trait dynamics correlation study dissects the gene regulation pattern in maize kernels.

BACKGROUND: Dissecting the genetic basis and regulatory mechanisms for the biosynthesis and accumulation of nutrients in maize could lead to the improved nutritional quality of this crop. Gene expression is regulated at the genomic, transcriptional, and post-transcriptional levels, all of which can produce diversity among traits. However, the expression of most genes connected with a particular trait usually does not have a direct association with the variation of that trait. In addition, expression profiles of genes involved in a single pathway may vary as the intrinsic cellular state changes. To work around these issues, we utilized a statistical method, liquid association (LA) to investigate the complex pattern of gene regulation in maize kernels. RESULTS: We applied LA to the expression profiles of 28,769 genes to dissect dynamic trait-trait correlation patterns in maize kernels. Among the 1000 LA pairs (LAPs) with the largest LA scores, 686 LAPs were identified conditional correlation. We also identified 830 and 215 LA-scouting leaders based on the positive and negative LA scores, which were significantly enriched for some biological processes and molecular functions. Our analysis of the dynamic co-expression patterns in the carotene biosynthetic pathway clearly indicated the important role of lcyE, CYP97A, ZEP1, and VDE in this pathway, which may change the direction of carotene biosynthesis by controlling the influx and efflux of the substrate. The dynamic trait-trait correlation patterns between gene expression and oil concentration in the fatty acid metabolic pathway and its complex regulatory network were also assessed. 23 of 26 oil-associated genes were correlated with oil concentration conditioning on 580 LA-scoutinggenes, and 5% of these LA-scouting genes were annotated as enzymes in the oil metabolic pathway. CONCLUSIONS: By focusing on the carotenoid and oil biosynthetic pathways in maize, we showed that a genome-wide LA analysis provides a novel and effective way to detect transcriptional regulatory relationships. This method will help us understand the biological role of maize kernel genes and will benefit maize breeding programs.

Correlations between ADC values and molecular markers of Ki-67 and HIF-1α in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths. Cell proliferativity and hypoxia have important impact on the response to radiotherapy or chemotherapy. The purpose of this study was to investigate the association of apparent diffusion coefficient (ADC) values and the molecular markers Ki-67 and hypoxia inducible factor-α (HIF-α) in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Forty-seven patients diagnosed with HCC were included in this study. All patients performed diffusion-weighted magnetic resonance imaging (DW-MRI) before any anticancer treatment. The ADC maps were automatically calculated on a Syngo workstation. The Ki-67 and HIF-1α expression were assessed by immunohistochemistry. The Pearson correlation test was used to assess the correlation between ADC values and Ki-67 and HIF-1α expression. RESULTS: Ki-67 staining was clearly identified based on the brown nuclear staining in tumor cells. High Ki-67 expression was correlated with low differentiation (p=0.028). A significant correlation was observed between HIF-1α expression and maximum diameter (p=0.014). The mean ADC value was (0.983±0.21)×10(-3) mm(2)/s. The level of Ki-67 expression was correlated inversely with the ADC values (r=-0.371, p=0.01). There was a significant positive correlation between the ADC values and HIF-1α expression (r=0.389, p=0.007). CONCLUSION: The ADC values were observed to correlate significantly with the molecular markers Ki-67 and HIF-1α. Our results suggest that the ADC values on DW-MRI may be used as a measure of cell proliferativity and hypoxia in hepatocellular carcinoma.

MR imaging in hepatocellular carcinoma: correlations between MRI features and molecular marker VEGF.

The aim of this paper was to investigate the correlations between magnetic resonance imaging (MRI) features and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC). Thirty-six patients with HCC included in this study underwent dynamic contrast-enhanced MR imaging and diffusion-weighted MR imaging. Signal intensity characteristics of HCCs were reviewed independently by two experienced radiologists. ADC maps were automatically computed. VEGF expression was evaluated by immunohistochemical staining. The grades of the VEGF expression were correlated inversely with the ADC values of the HCCs (r = -0.435, p = 0.008). Intensity heterogeneity correlated with VEGF expression (r = 0.571, p = 0.039). There is a significant correlation between the intensity of arterial enhancement and VEGF expression (r = -0.386, p = 0.02). However, no correlation was found between the VEGF grades and the intensity of enhancement in the hepatic portal venous and late phases, as well as equilibrium phase. Our results indicate that ADC value on DW-MRI, the signal intensity in arterial phase and intensity heterogeneity correlate with the degree of VEGF expression in hepatocellular carcinomas.

Functional investigations on embryonic stem cells labeled with clinically translatable iron oxide nanoparticles.

Stem cell based therapies offer significant potential in the field of regenerative medicine. The development of superparamagnetic iron oxide (SPIO) nanoparticle labeling and magnetic resonance imaging (MRI) have been increasingly used to track the transplanted cells, enabling in vivo determination of cell fate. However, the impact of SPIO-labeling on the cell phenotype and differentiation capacity of embryonic stem cells (ESCs) remains unclear. In this study, we wrapped SPIO nanoparticles with stearic acid grafted PEI600, termed as Stearic-LWPEI-SPIO, to generate efficient and non-toxic ESC labeling tools. Our results showed that efficient labeling of ESCs at an optimized low dosage of Stearic-LWPEI-SPIO nanoparticles did not alter the differentiation and self-renewal properties of ESCs. The localization of the transplanted ESCs observed by MRI correlated well with histological studies. These findings demonstrate that Stearic-LWPEI-SPIO nanoparticles have potential to be clinically translatable MRI probes and may enable non-invasive in vivo tracking of ESCs in experimental and clinical settings during cell-based therapies.

Evaluation of two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods for the analysis of baclofen and gabapentin in feeds by liquid chromatography tandem mass spectrometry.

This paper presents a new analytical method for the simultaneous determination of baclofen and gabapentin in feeds based on two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods and liquid chromatography tandem mass spectrometry (LC-MS/MS). For the two modified QuEChERS methods, samples were first extracted with acidified acetonitrile (5.0% acetic acid, v/v) without using acetonitrile salting-out extraction. Then, the first modified QuEChERS method was established according to the original QuEChERS cleanup procedure. For the second modified QuEChERS method, the extract was evaporated to dryness and reconstituted in acetonitrile. Subsequently, the analytes in the reconstituted solution were retained by primary secondary amine (PSA) and released from PSA with 1.0% formic acid in methanol. Finally, the eluate was evaporated and dissolved in 0.1% formic acid solution/methanol (v/v, 80:20). All of the samples were analyzed by LC-MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as the mobile phase with gradient elution. The matrix effect, recovery, and repeatability, within laboratory reproducibility, and the LODs and LOQs of the two modified QuEChERS sample preparation methods were investigated and compared. Comparative results showed that the second method was obviously superior to the first method.

Berberine potently attenuates intestinal polyps growth in ApcMin mice and familial adenomatous polyposis patients through inhibition of Wnt signalling.

As a traditional anti-inflammatory Chinese herbal medicine, Alkaloid berberine has been recently reported to exhibit anti-tumour effects against a wide spectrum of cancer. However, the mechanism was largely unknown. Gene chip array reveals that with berberine treatment, c-Myc, the target gene of Wnt pathway, was down-regulated 5.3-folds, indicating that berberine might inhibit Wnt signalling. TOPflash analysis revealed that Wnt activity was significantly reduced after berberine treatment, and the mechanism of which might be that berberine disrupted ß-catenin transfer to nucleus through up-regulating the expression of adenomatous polyposis coli (APC) gene and stabilized APC-ß-catenin complex. Berberine administration in ApcMin/+ mice exhibited fewer and smaller polyps in intestine, along with reduction in cyclin D1 and c-Myc expression. In clinical practice, oral administration of berberine also significantly reduced the familial adenomatous polyposis patients' polyp size along with the inhibition of cyclin D1 expression in polyp samples. These observations indicate that berberine inhibits colon tumour formation through inhibition of Wnt/ß-catenin signalling and berberine might be a promising drug for the prevention of colon cancer.

Simultaneous determination of six resorcylic acid lactones in feed using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a dispersive solid phase extraction sorbent.

A simple and cost-effective pre-treatment procedure was developed for six resorcylic acid lactones (RALs) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra-high performance liquid chromatography-negative electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). After extraction with acetonitrile/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, the extraction time for MWCNTs, the type and amount of MWCNTs and the type of eluent were optimised to increase the sample throughput and the sensitivity. The samples were quantified using the internal standard zearalenone-D6. The absolute recoveries of the target compounds from feed samples were most efficient when using 100mg of MWCNTs with an outer diameter of less than 8nm and a length of 10-30µm, and ethyl acetate was shown to be the most suitable solvent for desorbing the target compounds from the MWCNTs. The mean recoveries from fortified swine mixed feed samples ranged from 95.3% to 107.2% and had relative standard deviations lower than 10%; the limits of detection and quantification for RALs were in the ranges of 0.20-0.29µg/kg and 0.54-0.78µg/kg, respectively.

Development and validation of an ultra high performance liquid chromatography tandem mass spectrometry method for determination of 10 cephalosporins and desacetylcefapirin in milk.

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 10 cephalosporins and desacetylcefapirin in bovine milk by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Samples were directly purified through HLB cartridge after dilution with 50mM phosphate buffer solution (pH 8.5). Then the eluate was dried under nitrogen and the residue was redissolved in mobile phase. Samples were analyzed by LC-MS/MS on an Acquity UPLC BEH Shield RP18 column with gradient elution. The samples were quantified using ceftiofur-D3 as internal standard. The proposed method was validated according to the European Commission Decision 2002/657/EC. The CCα values were 111, 0.04, 140, 55, 55, 67, 23, 23, 68, 0.10 and 113µg/kg for cefalexin, cefradine, cefacetrile, cefazolin, cefoperazone, cefapirin, cefalonium, cefquinome, desacetylcefapirin, cefotaxime and ceftiofur, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CVr), and reproducibility (CVR) varied from 94.6% to 117.1%, from 5.6% to 13.6% (CVr), and from 5.9% to 27.9% (CVR), respectively. The method is demonstrated to be suitable for the determination of 10 cephalosporins and desacetylcefapirin in bovine milk. The total time required for the analysis of one sample, including sample preparation, was about 40min.

MicroRNA-24 regulates cardiac fibrosis after myocardial infarction.

Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure. Although dysregulation of microRNA (miRNA) is involved in various pathophysiological processes in the heart, the role of miRNA in fibrosis regulation after MI is not clear. Previously we observed the correlation between fibrosis and the miR-24 expression in hypertrophic hearts, herein we assessed how miR-24 regulates fibrosis after MI. Using qRT-PCR, we showed that miR-24 was down-regulated in the MI heart; the change in miR-24 expression was closely related to extracellular matrix (ECM) remodelling. In vivo, miR-24 could improve heart function and attenuate fibrosis in the infarct border zone of the heart two weeks after MI through intramyocardial injection of Lentiviruses. Moreover, in vitro experiments suggested that up-regulation of miR-24 by synthetic miR-24 precursors could reduce fibrosis and also decrease the differentiation and migration of cardiac fibroblasts (CFs). TGF-ß (a pathological mediator of fibrotic disease) increased miR-24 expression, overexpression of miR-24 reduced TGF-ß secretion and Smad2/3 phosphorylation in CFs. By performing microarray analyses and bioinformatics analyses, we found furin to be a potential target for miR-24 in fibrosis (furin is a protease which controls latent TGF-ß activation processing). Finally, we demonstrated that protein and mRNA levels of furin were regulated by miR-24 in CFs. These findings suggest that miR-24 has a critical role in CF function and cardiac fibrosis after MI through a furin-TGF-ß pathway. Thus, miR-24 may be used as a target for treatment of MI and other fibrotic heart diseases.

Insulin redirects differentiation from cardiogenic mesoderm and endoderm to neuroectoderm in differentiating human embryonic stem cells.

Human embryonic stem cells (hESC) can proliferate indefinitely while retaining the capacity to form derivatives of all three germ layers. We have reported previously that hESC differentiate into cardiomyocytes when cocultured with a visceral endoderm-like cell line (END-2). Insulin/insulin-like growth factors and their intracellular downstream target protein kinase Akt are known to protect many cell types from apoptosis and to promote proliferation, including hESC-derived cardiomyocytes. Here, we show that in the absence of insulin, a threefold increase in the number of beating areas was observed in hESC/END-2 coculture. In agreement, the addition of insulin strongly inhibited cardiac differentiation, as evidenced by a significant reduction in beating areas, as well as in alpha-actinin and beta-myosin heavy chain (beta-MHC)-expressing cells. Real-time reverse transcription-polymerase chain reaction and Western blot analysis showed that insulin inhibited cardiomyogenesis in the early phase of coculture by suppressing the expression of endoderm (Foxa2, GATA-6), mesoderm (brachyury T), and cardiac mesoderm (Nkx2.5, GATA-4). In contrast to previous reports, insulin was not sufficient to maintain hESC in an undifferentiated state, since expression of the pluripotency markers Oct3/4 and nanog declined independently of the presence of insulin during coculture. Instead, insulin promoted the expression of neuroectodermal markers. Since insulin triggered sustained phosphorylation of Akt in hESC, we analyzed the effect of an Akt inhibitor during coculture. Indeed, the inhibition of Akt or insulin-like growth factor-1 receptor reversed the insulin-dependent effects. We conclude that in hESC/END-2 cocultures, insulin does not prevent differentiation but favors the neuroectodermal lineage at the expense of mesendodermal lineages.

Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations > or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.