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The conductance of K ATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface K ATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.

Recent studies indicate that both genomic alterations and transcriptional dysregulation influence the disease progresses. This study proposes a method identifying pathways by integrating copy numbers (CN), gene expressions (GE) and their correlations. A lung cancer patients dataset with both normal and tumor tissues is utilized to evaluate the performance of the proposed method. To further appraise the predicting abilities of those pathways, these patients are classified by support vector machines. Based on the classification results, pathways integrating CN, GE and their correlations is more informative and biologically meaningful and perform better than pathways obtained by only CN or only GE.

Hydrogen sulfide (H(2)S), as an endogenous gas signaling molecule with important biological function that has been found recently, may play a protection in ischemic reperfusion (I/R) myocardium. We investigated the cardioprotective effect of H(2)S in rats model of ischemic reperfusion in vivo and a probably influence on the expression of survivin, an anti-apoptosis gene. Animals were randomly divided into 3 groups and received either vehicle, sodium hydrosulfide (NaHS) or DL-propargylglycine (PAG) respectively everyday for 1 week before surgery and the treatment continued for a further 2 d after I/R till the animals were sacrificed. We investigated the plasma H(2)S concentration and blood pressure, with the electrocardiogram (ECG) together, to prove the effect of H(2)S to the heart function. We also compared the heart infarct size and the expression of an anti-apoptosis gene, survivin, among groups. As the data shown, the NaHS group had great improvement in blood pressure and electrocardiogram situation. And the remarkable shrink of the infarct size and up-regulation of survivin in NaHS group comparing with the other two groups also showed the cardio protective effect of H(2)S in our study.

Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity and change from a quiescent contractile phenotype to a proliferative synthetic phenotype during physiological arteriogenesis and pathological conditions such as atherosclerosis and restenosis. Platelet-derived growth factor (PDGF)-BB is a potent inducer of the VSMC synthetic phenotype; however, much less is known about the role of fibroblast growth factor-2 (FGF2) in this process. Here, we show using signal transduction mutants of FGF receptor 1 (FGFR1) expressed in rat VSMC that the adaptor protein FRS2 is essential for FGFR1-mediated phenotypic modulation and down-regulation of VSMC smooth muscle alpha-actin (SMA) gene expression. In addition, we show that PDGF-BB and FGF2 act synergistically to induce cell proliferation and down-regulate SMA and SM22alpha in VSMC. Furthermore, we show that PDGF-BB induces tyrosine phosphorylation of FGFR1 and that this phosphorylation is mediated by PDGF receptor-beta (PDGFRbeta), but not c-Src. We demonstrate that FRS2 co-immunoprecipitates with PDGFRbeta in a complex that requires FGFR1 and that both the extracellular and the intracellular domains of FGFR1 are required for association with PDGFRbeta, whereas the cytoplasmic domain of FGFR1 is required for FRS2 association with the FGFR1-PDGFRbeta complex. Knockdown of FRS2 in VSMC by RNA interference inhibited PDGF-BB-mediated down-regulation of SMA and SM22alpha without affecting PDGF-BB mediated cell proliferation or ERK activation. Together, these data support the notion that PDGFRbeta down-regulates SMA and SM22alpha through formation of a complex that requires FGFR1 and FRS2 and prove novel insight into VSMC phenotypic plasticity.

Synthesized gene of human thymosin alpha 1 (Talpha1) was inserted into pET-28a, pET-9c, pThioHis B, pGEX-2T or pBV222 and then inductively expressed in strains of Escherichia coli. Among the five expression systems, the BL21/pET-28a system provides the highest expression level of fusion protein in a soluble form, which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting fusion protein purified through nickel affinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity by SDS-PAGE. These results indicate the potential of this expression system for high-throughput production of recombinant Talpha1.

Simple Summary: The elongation of very-long-chain fatty acids gene 2 (elovl2) of Chinese sturgeon was identified in acipenseriformes species for the first time. Elovl2 was highly conserved in molecular evolution among vertebrates. Functional characterization in yeast demonstrated that Chinese sturgeon Elovl2 could efficiently elongate C20 (20:4n-6 and 20:5n-3) and C22 (22:4n-6 and 22:5n-3) substrates, confirming its critical roles in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. Diets with different types of PUFAs can affect the transcription levels of elovl2 in Chinese sturgeon. This study enhances our understanding about the evolutionary and functional characteristics of elovl2 and also provides novel insights into the mechanism of LC-PUFA biosynthesis in vertebrates. Elongases of very-long-chain fatty acids (Elovls) are critical rate-limiting enzymes that are involved in LC-PUFA biosynthesis through catalyzing the two-carbon elongation of a pre-existing fatty acyl chain. Thus far, several Elovls have been extensively studied in teleost. However, the functional and physiological roles of Elovls in chondrichthyans have rarely been reported. In this study, we identified and characterized elovl2 from the endangered Chinese sturgeon (Acipenser sinensis) by whole genome scanning. The results show that the coding sequence of elovl2 was 894 bp in length, for a putative protein of 297 amnio acids. Comparative genomic analyses indicated that Chinese sturgeon elovl2 was evolutionarily conserved. Functional characterization in yeast demonstrated that the Chinese sturgeon Elovl2 could efficiently elongate C20 (ARA and EPA) and C22 (22:4n-6 and 22:5n-3) substrates, confirming its critical roles in LC-PUFA biosynthesis. Spatial and temporal expression analyses showed high elovl2 mRNA levels were detected in the liver and brain and showed an increase trend both in embryonic and post-hatching stages. Interestingly, diets with vegetable oils as lipid sources could significantly induce the high expression of elovl2 in Chinese sturgeon, implying that the endogenous LC-PUFA biosynthesis pathway was stimulated by lack of LC-PUFA in their diets. Our findings will enhance our understanding about the evolutionary and functional roles of elovl2 and provide novel insights into the LC-PUFA biosynthesis mechanism in vertebrates. [ABSTRACT FROM AUTHOR]

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is characterized by high mortality and morbidity rates. Vav guanine nucleotide exchange factors (VAVs), crucial for signal transduction between cell membrane receptors and intracellular mediators, have been implicated in carcinogenesis. However, their potential prognostic value in RCC remains unclear. The impact of 150 common VAV polymorphisms on RCC risk and survival was investigated in a cohort of 630 individuals. Publicly available gene expression datasets were utilized to analyze VAV gene expression in relation to patient outcomes. The VAV3 rs17019888 polymorphism was significantly associated with RCC risk and overall survival after adjusting for false discovery rates. Expression quantitative trait loci analysis revealed that the risk allele of rs17019888 is linked to reduced VAV3 expression. Analysis of 19 kidney cancer gene expression datasets revealed lower VAV3 expression in RCC tissues compared to normal tissues, with higher expression correlating with better prognosis. Gene set enrichment analysis demonstrated that VAV3 negatively regulates the ubiquitin–proteasome system, extracellular matrix and membrane receptors, inflammatory responses, matrix metalloproteinases, and cell cycle pathways. Furthermore, elevated VAV3 expression was associated with increased infiltration of B cells, macrophages, and neutrophils into the RCC tumor microenvironment. Our findings suggest that VAV3 gene variants influence RCC risk and survival, contributing to a favorable prognosis in RCC. [ABSTRACT FROM AUTHOR]

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication. [ABSTRACT FROM AUTHOR]

Background: Childhood allergies of asthma and atopic dermatitis (AD) involve an overactive T‐cell immune response triggered by allergens. However, the impact of T‐cell receptor (TCR) repertoires on allergen sensitization and their role in mediating different phenotypes of asthma and AD in early childhood remains unclear. Methods: A total of 78 children, comprising 26 with asthma alone, 26 with AD alone, and 26 healthy controls (HC), were enrolled. TCR repertoire profiles were determined using a unique molecular identifier system for next‐generation sequencing. Integrative analyses of their associations with allergen‐specific IgE levels and allergies were performed. Results: The diversity in TCR alpha variable region (TRAV) genes of TCR repertoires and complementarity determining region 3 (CDR3) clonality in TRAV/TRBV (beta) genes were significantly higher in children with AD compared with those with asthma and HC (p <.05). Compared with HC, the expression of TRAV13‐1 and TRAV4 genes was significantly higher in both asthma and AD (p <.05), with a significant positive correlation with mite‐specific IgE levels (p <.01). In contrast, TRBV7‐9 gene expression was significantly lower in both asthma and AD (p <.01), with this gene showing a significant negative correlation with mite‐specific IgE levels (p <.01). Furthermore, significantly higher TRAV8‐3 gene expression, positively correlated with food‐specific IgE levels, was found in children with AD compared with those with asthma (p <.05). Conclusion: Integrated TCR repertoires analysis provides clinical insights into the diverse TCR genes linked to antigen specificity, offering potential for precision immunotherapy in childhood allergies. [ABSTRACT FROM AUTHOR]

Background: Disease progression in biosystems is not always a steady process but is occasionally abrupt. It is important but challenging to signal critical transitions in complex biosystems. Methods: In this study, based on the theoretical framework of dynamic network biomarkers (DNBs), we propose a model-free method, edge-based relative entropy (ERE), to identify temporal key biomolecular associations/networks that may serve as DNBs and detect early-warning signals of the drastic state transition during disease progression in complex biological systems. Specifically, by combining gene‒gene interaction (edge) information with the relative entropy, the ERE method converts gene expression values into network entropy values, quantifying the dynamic change in a biomolecular network and indicating the qualitative shift in the system state. Results: The proposed method was validated using simulated data and real biological datasets of complex diseases. The applications show that for certain diseases, the ERE method helps to reveal so-called "dark genes" that are non-differentially expressed but with high ERE values and of essential importance in both gene regulation and prognosis. Conclusions: The proposed method effectively identified the critical transition states of complex diseases at the network level. Our study not only identified the critical transition states of various cancers but also provided two types of new prognostic biomarkers, positive and negative edge biomarkers, for further practical application. The method in this study therefore has great potential in personalized disease diagnosis. [ABSTRACT FROM AUTHOR]

Background: Endometrial fibrosis, a significant characteristic of intrauterine adhesion (IUA), is caused by the excessive differentiation and activation of endometrial stromal cells (ESCs). Glutaminolysis is the metabolic process of glutamine (Gln), which has been implicated in multiple types of organ fibrosis. So far, little is known about whether glutaminolysis plays a role in endometrial fibrosis. Methods: The activation model of ESCs was constructed by TGF-β1, followed by RNA-sequencing analysis. Changes in glutaminase1 (GLS1) expression at RNA and protein levels in activated ESCs were verified experimentally. Human IUA samples were collected to verify GLS1 expression in endometrial fibrosis. GLS1 inhibitor and glutamine deprivation were applied to ESCs models to investigate the biological functions and mechanisms of glutaminolysis in ESCs activation. The IUA mice model was established to explore the effect of glutaminolysis inhibition on endometrial fibrosis. Results: We found that GLS1 expression was significantly increased in activated ESCs models and fibrotic endometrium. Glutaminolysis inhibition by GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES or glutamine deprivation treatment suppressed the expression of two fibrotic markers, α-SMA and collagen I, as well as the mitochondrial function and mTORC1 signaling in ESCs. Furthermore, inhibition of the mTORC1 signaling pathway by rapamycin suppressed ESCs activation. In IUA mice models, BPTES treatment significantly ameliorated endometrial fibrosis and improved pregnancy outcomes. Conclusion: Glutaminolysis and glutaminolysis-associated mTOR signaling play a role in the activation of ESCs and the pathogenesis of endometrial fibrosis through regulating mitochondrial function. Glutaminolysis inhibition suppresses the activation of ESCs, which might be a novel therapeutic strategy for IUA. [ABSTRACT FROM AUTHOR]

Simple Summary: Endoparasitic wasps are nature's pest controllers, targeting various harmful insects. They lay their eggs inside these pests and release special substances (like viruses or venoms) that change the way the pest's immune system works. However, we are still uncovering exactly how these changes happen. Our research looks into how certain wasps, which do not use these viruses, can still affect their host's immune system. We studied this using a specific wasp–beetle pair as an example. We found that these wasps can control a crucial part of the beetle's immune response through a process that modifies the beetle's genetic material, specifically by adjusting certain markers on the genes that are important for immunity. This discovery sheds light on the sophisticated ways wasps can suppress the immune systems of their hosts, making them more effective at controlling pest populations. Endoparasitoids are insects that develop within other insects, employing unique strategies to enhance their offspring's survival. They inject polydnavirus and/or venom into their hosts along with eggs, effectively suppressing the host's immune system. Polydnavirus from Braconidae and Ichneumonidae wasps can integrate into the host's genome to express viral genes using the host's transcription systems. However, the ability of parasitoids without polydnavirus to manipulate host gene expression remains unclear. Lysine acetylation (LysAc), a post-translational modification critical for gene regulation, is hypothesized to be used by endoparasitoids lacking polydnavirus. We utilized the Chalcidoidea wasp Tetrastichus brontispae, which lacks polydnavirus, as an idiobiont endoparasitoid model to test this hypothesis, with pupae of the nipa palm hispid beetle Octodonta nipae as the host. Parasitism by T. brontispae resulted in the reduced expression of histone deacetylase Rpd3 and elevated levels of LysAc modification at histones H3.3K9 and H3.3K14 through proteomics and LysAc modification omics. The knockdown of Rpd3 increased the expression level of OnPPAF1 and OnPPO involved in the phenoloxidase cascade, leading to melanization in the host body whereby it resembled a mummified parasitized pupa and ultimately causing pupa death. This study enhances our understanding of how endoparasitoids employ histone acetylation to regulate immunity-related genes, offering valuable insights into their survival strategies. [ABSTRACT FROM AUTHOR]

The critical point or pivotal threshold of cell transition occurs in early embryonic development when cell differentiation culminates in its transition to specific cell fates, at which the cell population undergoes an abrupt and qualitative shift. Revealing such critical points of cell transitions can track cellular heterogeneity and shed light on the molecular mechanisms of cell differentiation. However, precise detection of critical state transitions proves challenging when relying on single-cell RNA sequencing data due to their inherent sparsity, noise, and heterogeneity. In this study, diverging from conventional methods like differential gene analysis or static techniques that emphasize classification of cell types, an innovative computational approach, single-cell gene association entropy (SGAE), is designed for the analysis of single-cell RNA-seq data and utilizes gene association information to reveal critical states of cell transitions. More specifically, through the translation of gene expression data into local SGAE scores, the proposed SGAE can serve as an index to quantitatively assess the resilience and critical properties of genetic regulatory networks, consequently detecting the signal of cell transitions. Analyses of five single-cell datasets for embryonic development demonstrate that the SGAE method achieves better performance in facilitating the characterization of a critical phase transition compared with other existing methods. Moreover, the SGAE value can effectively discriminate cellular heterogeneity over time and performs well in the temporal clustering of cells. Besides, biological functional analysis also indicates the effectiveness of the proposed approach. [ABSTRACT FROM AUTHOR]

Background: Endothelial-mesenchymal transition (EndoMT) is an emerging adaptive process that modulates lymphatic endothelial function to drive aberrant lymphatic vascularization in the tumour microenvironment (TME); however, the molecular determinants that govern the functional role of EndoMT remain unclear. Here, we show that cancer-associated fibroblast (CAF)-derived PAI-1 promoted the EndoMT of lymphatic endothelial cells (LECs) in cervical squamous cell carcinoma (CSCC). Methods: Immunofluorescent staining of α-SMA, LYVE-1 and DAPI were examined in primary tumour samples obtained from 57 CSCC patients. Assessment of cytokines secreted by CAFs and normal fibroblasts (NFs) was performed using human cytokine antibody arrays. The phenotype of EndoMT in lymphatic endothelial cells (LECs), gene expression levels, protein secretion and activity of signaling pathways were measured by real-time RT-PCR, ELISA or western blotting. The function of lymphatic endothelial monolayers was examined by transwell, tube formation assay, transendothelial migration assay in vitro. Lymphatic metastasis was measured using popliteal lymph node metastasis model. Furthermore, association between PAI-1 expression and EndoMT in CSCC was analyzed by immunohistochemistry. The Cancer Genome Atlas (TCGA) databases was used to assess the association of PAI-1 with survival rate in CSCC. Results: CAF-derived PAI-1 promoted the EndoMT of LECs in CSCC. LECs undergoing EndoMT could initiate tumour neolymphangiogenesis that facilitated cancer cell intravasation/extravasation, which in turn promoted lymphatic metastasis in CSCC. Mechanistically, PAI-1 activated the AKT/ERK1/2 pathways by directly interacting with low-density lipoprotein receptor-related protein (LRP1), thereby leading to elevated EndoMT activity in LECs. Blockade of PAI-1 or inhibition of LRP1/AKT/ERK1/2 abrogated EndoMT and consequently attenuated CAF-induced tumour neolymphangiogenesis. Furthermore, clinical data revealed that increased PAI-1 levels positively correlated with EndoMT activity and poor prognosis in CSCC patients. Conclusion: Our data indicate that CAF-derived PAI-1 acts as an important neolymphangiogenesis-initiating molecular during CSCC progression through modulating the EndoMT of LECs, resulting in promotion of metastasis ability in primary site. PAI-1 could serve as an effective prognostic biomarker and therapeutic target for CSCC metastasis. [ABSTRACT FROM AUTHOR]

Objective: Dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) are collectively known as Lewy body dementia (LBD). Considering the heterogeneous nature of LBD and the different constellations of symptoms with which patients can present, the exact molecular mechanism underlying the differences between these two isoforms is still unknown. Therefore, this study aimed to explore the biomarkers and potential mechanisms that distinguish between PDD and DLB. Methods: The mRNA expression profile dataset of GSE150696 was acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between 12 DLB and 12 PDD were identified from Brodmann area 9 of human postmortembrains using GEO2R. A series of bioinformaticsmethods were applied to identify the potential signaling pathways involved, and a protein-protein interaction (PPI) network was constructed. Weighted gene co-expression network analysis (WGCNA) was used to further investigate the relationship between gene co-expression and different LBD subtypes. Hub genes that are strongly associated with PDD and DLB were obtained from the intersection of DEGs and selected modules by WGCNA. Results: A total of 1,864 DEGs between PDD and DLB were filtered by the online analysis tool GEO2R. We found that the most significant GO- and KEGG-enriched terms are involved in the establishment of the vesicle localization and pathways of neurodegeneration-multiple diseases. Glycerolipid metabolism and viral myocarditis were enriched in the PDD group. A B-cell receptor signaling pathway and one carbon pool by folate correlated with DLB in the results obtained from the GSEA. We found several clusters of co-expressed genes which we designated by colors in our WGCNA analysis. Furthermore, we identified seven upregulated genes, namely, SNAP25,GRIN2A,GABRG2,GABRA1,GRIA1, SLC17A6, and SYN1, which are significantly correlated with PDD. Conclusion: The seven hub genes and the signaling pathways we identified may be involved in the heterogeneous pathogenesis of PDD and DLB. [ABSTRACT FROM AUTHOR]

Increasing adipogenesis has been explored to treat metabolic diseases and atherosclerosis through the release of adiponectin. The effects and mechanism of platelet-rich plasma treatment on fat graft survival and adipogenesis have not been clarified. Here, we aimed to study the effects of monocyte chemoattractant protein-1 (MCP-1)-supplemented plasma on adipogenesis-related gene expression and adiponectin levels. Stromal vascular fractions (SVFs) purified from the inguinal adipose tissue of obese and diabetic (Leprdb/db) mice were treated with plasma from control (Lepr+/+) mice supplemented with 10 or 50 ng of MCP-1. The expression of adiponectin and interleukin-33 (IL-33) mRNA in adipose tissue was increased in Leprdb/db mice, whereas control (Lepr+/+) plasma reduced expression of IL-33 mRNA as well as peroxisome proliferator-activated receptor gamma (PPARγ), pJNK, and pNF-κB protein, and increased the expression of IL-10 mRNA in SVFs of Leprdb/db mice. MCP-1-supplemented control plasma increased the expression of adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), dipeptidyl peptidase 4 (DPP4), IL-33, and PDGFα mRNA and the expression of adiponectin protein as well as PPARγ of SVFs and the expression of PPARγ mRNA in adipose tissue macrophages (ATMs). Injection of MCP-1-supplemented plasma into adipose tissue of Leprdb/db mice increased the expression of IL-33 and Col3a1 mRNA in SVFs and IL-33, FABP4, PDGFα, PPARγ and PPARγ2 of ATMs, protein expression of adiponectin and PPARγ of SVFs, and plasma adiponectin levels, as well as DPP4 activity. In conclusion, our results demonstrate that control plasma decreases adipogenesis and increases IL-10, and decreases IL-33, pJNK, and pNF-κB in adipose tissue. MCP-1-supplemented plasma enhances adipogenesis-related gene expression in SVFs and adiponectin levels, which may be mediated through an increase of IL-33 and PPARγ. Thus, our findings suggest that MCP-1-supplemented plasma represents a novel therapy to stimulate local adipogenesis and systemic adiponectin levels. [ABSTRACT FROM AUTHOR]

Cervical cancer has a poor prognosis and is the fourth most common cancer among women. Dihydromyricetin (DHM), a flavonoid compound, exhibits several pharmacological activities, including anticancer effects; however, the effects of DHM on cervical cancer have received insufficient research attention. This study examined the antitumor activity and underlying mechanisms of DHM on human cervical cancer. Our results indicated that DHM inhibits migration and invasion in HeLa and SiHa cell lines. Mechanistically, RNA sequencing analysis revealed that DHM suppressed S100A4 mRNA expression in HeLa cells. Moreover, DHM inhibited the protein expressions of β-catenin and GSK3β through the regulated extracellular-signal-regulated kinase (ERK)1/2 signaling pathway. By using the ERK1/2 activator, T-BHQ, reverted β-catenin and S100A4 protein expression and cell migration, which were reduced in response to DHM. In conclusion, our study indicated that DHM inhibited cell migration by reducing the S100A4 expression through the ERK1/2/β-catenin pathway in human cervical cancer cell lines. [ABSTRACT FROM AUTHOR]

The dynamics of complex diseases are not always smooth; they are occasionally abrupt, i.e. there is a critical state transition or tipping point at which the disease undergoes a sudden qualitative shift. There are generally a few significant differences in the critical state in terms of gene expressions or other static measurements, which may lead to the failure of traditional differential expression-based biomarkers to identify such a tipping point. In this study, we propose a computational method, the direct interaction network-based divergence, to detect the critical state of complex diseases by exploiting the dynamic changes in multivariable distributions inferred from observable samples and local biomolecular direct interaction networks. Such a method is model-free and applicable to both bulk and single-cell expression data. Our approach was validated by successfully identifying the tipping point just before the occurrence of a critical transition for both a simulated data set and seven real data sets, including those from The Cancer Genome Atlas and two single-cell RNA-sequencing data sets of cell differentiation. Functional and pathway enrichment analyses also validated the computational results from the perspectives of both molecules and networks. [ABSTRACT FROM AUTHOR]

Programmed death‐ligand 1 (PD‐L1), an immune checkpoint ligand, is recognized as a potential target for cancer immunotherapy as well as for the induction of transplantation tolerance. However, how the crosstalk between stem cell programming and cytokine signaling regulates PD‐L1 expression during stem cell differentiation and cancer cell plasticity remains unclear. Herein, we reported that PD‐L1 expression was regulated by SOX2 during embryonic stem cell (ESC) differentiation and lung cancer cell plasticity. PD‐L1 was induced during ESC differentiation to fibroblasts and was downregulated during SOX2‐mediated reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Furthermore, SOX2 activation affected cancer cell plasticity and inhibited PD‐L1 expression in lung cancer cells. We discovered that the H3K27ac signal at the PD‐L1 locus was enhanced during ESC differentiation to fibroblasts as well as during cancer plasticity of SOX2‐positive lung cancer cells to SOX2‐negative counterparts. Romidepsin, an epigenetic modifier, induced PD‐L1 expression in lung cancer cells, whereas TGF‐β stimulation downregulated SOX2 but upregulated PD‐L1 expression in lung cancer cells. Furthermore, in addition to PD‐L1, the expressions of EGFR and its ligand HBEGF were downregulated by activation of endogenous SOX2 expression during lung cancer cell plasticity and iPSC reprogramming, and the activation of EGFR signaling by HBEGF upregulated PD‐L1 expression in lung cancer cells. Together, our results reveal the crosstalk between SOX2 programming and cytokine stimulation influences PD‐L1 expression, and these findings may provide insights into PD‐L1‐mediated therapeutics. [ABSTRACT FROM AUTHOR]

Objective. The study is aimed at investigating the regulatory relationship between miR-145-5p and ABRACL, and has tried at clarifying the mechanisms underlying the proliferation, migration, and invasion of esophageal carcinoma (EC) cells. Methods. Gene expression data related to EC were accessed from TCGA database, and the "edgeR" package was used to screen differentially expressed genes. TargetScan, miRDB, and miRTarBase databases were used to predict potential targets for the target miRNA miR-145-5p. qRT-PCR and Western blot were performed to assess the expression of miR-145-5p and ABRACL in EC cells. Dual-luciferase reporter assay was performed to validate the targeting relationship between miR-145-5p and ABRACL. Functional experiments including CCK-8 assay, Transwell migration, and invasion assays were used to detect the proliferation, migration, and invasion of EC cells. Results. The expression of miR-145-5p was significantly decreased in EC, while ABRACL was remarkably increased. In addition, there was a negative correlation identified between miR-145-5p and ABRACL mRNA. Overexpressing miR-145-5p was able to suppress cell proliferation, migration, and invasion, whereas silencing miR-145-5p posed an opposite effect. In the meantime, ABRACL was identified as a direct target of miR-145-5p by dual-luciferase reporter assay. Furthermore, miR-145-5p could inhibit the expression of ABRACL, in turn inhibiting the proliferation, migration, and invasion of EC cells. Conclusion. miR-145-5p functions on the proliferation, migration, and invasion of EC cells via targeting ABRACL, and it may be a novel therapeutic target in EC treatment. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) help plants respond to environmental stresses by regulating gene expression. Up till now, studies on the MYB family of TFs have mainly focused on the highly abundant R2R3-subtype. While the less well-known 1R-subtype has been generally shown to enhance abscisic acid (ABA) sensitivity by acting as transcriptional activators, the mechanisms of their functions are unclear. Here we identified an ABA sensitivity-associated gene from soybean, ABA-Sensitive 1 (GmABAS1), of the 1R-subtype of MYB. Using the GFP-GmABAS1 fusion protein, we demonstrated that GmABAS1 is localized in the nucleus, and with yeast reporter systems, we showed that it is a transcriptional repressor. We then identified the target gene of GmABAS1 to be Glyma.01G060300 , an annotated ABI five-binding protein 3 and showed that GmABAS1 binds to the promoter of Glyma.01G060300 both in vitro and in vivo. Furthermore, Glyma.01G060300 and GmABAS1 exhibited reciprocal expression patterns under osmotic stress, inferring that GmABAS1 is a transcriptional repressor of Glyma.01G060300. As a further confirmation, AtAFP2 , an orthologue of Glyma.01G060300 , was down-regulated in GmABAS1 -transgenic Arabidopsis thaliana , enhancing the plant's sensitivity to ABA. This is the first time a 1R-subtype of MYB from soybean has been reported to enhance ABA sensitivity by acting as a transcriptional repressor. [ABSTRACT FROM AUTHOR]

Objectives The aim of our study was to ascertain the underlying role of microRNAs (miRNAs) in human intervertebral disc degeneration (IDD). Design Bioinformatic analysis from multiple databases. Methods Studies of the association of miRNAs and IDD were identified in multiple electronic databases. All potential studies were assessed by the same inclusion and exclusion criteria. We recorded whether miRNA expression was commonly increased or suppressed in the intervertebral disc tissues and cells of IDD subjects. We used String to identify biological process and cellular component pathways of differentially expressed genes. Results We included fifty-seven articles from 1,277 records in this study. This report identified 40 different dysregulated miRNAs in 53 studies, including studies examining cell apoptosis (26 studies, 49.06%), cell proliferation (15 studies, 28.3%), extracellular matrix (ECM) degradation (10 studies, 18.86%), and inflammation (five studies, 9.43%) in IDD patients. Three upregulated miRNAs (miR-19b, miR-32, miR-130b) and three downregulated miRNAs (miR-31, miR-124a, miR-127-5p) were considered common miRNAs in IDD tissues. The top three biological process pathways for upregulated miRNAs were positive regulation of biological process, nervous system development, and negative regulation of biological process, and the top three biological process pathways for downregulated miRNAs were negative regulation of gene expression, intracellular signal transduction, and negative regulation of biological process. Conclusions This study revealed that miRNAs could be novel targets for preventing IDD and treating patients with IDD by regulating their target genes. These results provide valuable information for medical professionals, IDD patients, and health care policy makers. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a disease characterized by abnormal cell proliferation in the bone marrow and is the most common quickly progressive leukemia in adults. Pinostrobin, a flavonoid phytochemical, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties. In this study, we aimed to investigate the antileukemic effects of pinostrobin and its molecular mechanisms in human AML cells. Our study found that pinostrobin (0–80 μM) significantly reduced the viability of human AML cells, with the pronounced cytotoxic effects observed in MV4-11 > MOLM-13 > HL-60 > U-937 > THP-1 cells. Pinostrobin was found to suppress leukemia cell proliferation, modulate cell cycle progression, promote cell apoptosis, and induce monocytic differentiation in MV4-11 cells. In animal studies, pinostrobin significantly suppressed the growth of leukemia cells in a zebrafish xenograft model. Microarray-based transcriptome analysis showed that the differentially expressed genes (DEGs) in pinostrobin-treated cells were strongly associated with enriched Gene Ontology (GO) terms related to apoptotic process, cell death, cell differentiation, cell cycle progression, and cell division. Combining DisGeNET and STRING database analysis revealed that pinostrobin upregulates forkhead box 3 (FOXO3), a tumor suppressor in cancer development, and plays an essential role in controlling AML cell viability. Our study demonstrated that pinostrobin increases FOXO3 gene expression and promotes its nuclear translocation, leading to the inhibition of cell growth. Finally, the study found that pinostrobin, when combined with cytarabine, synergistically reduces the viability of AML cells. Our current findings shed light on pinostrobin's mechanisms in inhibiting leukemia cell growth, highlighting its potential as a chemotherapeutic agent or nutraceutical supplement for AML prevention or treatment. • Pinostrobin demonstrates anti-leukemic effects in human AML cells in vitro and zebrafish xenografts in vivo. • Pinostrobin inhibits cell growth by elevating FOXO3 gene expression and prompting its nuclear accumulation in AML cells. • The combination of cytarabine and pinostrobin demonstrates a synergistic effect in reducing AML cell growth. • Pinostrobin could serve as a therapeutic agent or a nutraceutical supplement in conjunction with clinical drugs for AML. [ABSTRACT FROM AUTHOR]

Background. Panax notoginseng (Burk.) F. H. Chen is one of the most common herbs in China. Because of its good efficacy and little adverse reaction, Panax notoginseng has been used widely to treat cardiovascular diseases (CVDs). Objective. To investigate effects of Panax notoginseng flower (PN-F) on rats with myocardial infarction (MI). Methods. The proximal left anterior descending coronary artery in rats was ligated to induce acute myocardial infarction. Then, animals were randomly assigned to four experimental groups: MI control group, Betaloc control group (with Betaloc 10 mg/kg/d), FD500 (low-dose) group (Panax notoginseng flower decoction 500 mg/kg, n=10), and FD1000 (high-dose) group (Panax notoginseng flower decoction 1000 mg/kg, n=10). Panax notoginseng flower decoction or Betaloc was orally administrated for two to four weeks before and after operation. Sham-operated group was used as a normal untreated group, in which animals were treated with double distilled water, once daily. HE (hematoxylin and eosin) staining, immunofluorescent assay, TUNEL assay, quantitative real-time PCR, and western blot analysis were, respectively, performed to observe morphology, count mean minimal vessels, investigate apoptotic cells, and record gene (HIF-1, VEGFA, and KDR) and protein (Bcl-2 and Bax) expressions. Results. Two weeks after MI, PN-F significantly enhanced capillary density in the border area of MI, decreased infarct size, improved minimal vessels, suppressed cell apoptosis, and enhanced expressions of genes (HIF-1, VEGFA, and KDR) and proteins (Bcl-2 and Bax). Conclusions. PN-F demonstrated a potential herb to treat rats with myocardial infarction through promoting angiogenesis and inhibition of apoptosis in the infarct area. [ABSTRACT FROM AUTHOR]

The progression of complex diseases is generally divided as a normal state, a pre-disease state or tipping point, and a disease state. Developing individual-specific method that can identify the pre-disease state just before a catastrophic deterioration, is critical for patients with complex diseases. However, with only a case sample, it is challenging to detect a pre-disease state which has little significant differences comparing with a normal state in terms of phenotypes and gene expressions. In this study, by regarding the tipping point as the end point of a stationary Markov process, we proposed a single-sample-based hidden Markov model (HMM) approach to explore the dynamical differences between a normal and a pre-disease states, and thus can signal the upcoming critical transition immediately after a pre-disease state. Using this method, we identified the pre-disease state or tipping point in a numerical simulation and two real datasets including stomach adenocarcinoma and influenza infection, which demonstrate the effectiveness of the method. [ABSTRACT FROM AUTHOR]

Particulate matter (PM) is one of the most important environmental issues worldwide, which is associated with not only pulmonary but also cardiovascular diseases. However, the underlying biological mechanisms of PM related cardiovascular dysfunction remained poorly defined, especially mediated by the pathway of direct impact on vascular and heart. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an ideal platform for studying PM-exposed cellular diseases model in vitro. Here, we investigated the direct effects of particulate matter 2.5 (PM2.5) on hiPSC-CMs and the potential mechanism at non-cytotoxic concentrations. Cell viability, contraction amplitude and spontaneous beating rate of iPSC-CMs after direct exposure to PM2.5 showed that the concentration of lower than 100 µg/ml would not lead to cytotoxic effects. Calcium-mediated optical mapping illustrated that there was a concentration-dependent reduction in quantification of calcium transient amplitude and an increase in the incidence of early after depolarizations due to PM2.5 treatment. Furthermore, there were dramatic dosage-dependent shortening in action potential duration and decrease in L-type calcium peak current density. The Ingenuity Pathway Analysis of our transcriptive study indicated that PM2.5 exposure preferentially influenced the expression of genes involved in calcium signaling. Among them the up-regulation of TRPC3 potentially played an important role in the electrophysiological alteration of PM2.5 on hiPSC-CMs, which could be ameliorated by pretreatment with pyr3, the inhibitor of TRPC3. In conclusion, our results demonstrated that exposure to PM2.5 was capable of increasing propensity to cardiac arrhythmias which could be attenuated with TRPC3 inhibition. [ABSTRACT FROM AUTHOR]

In order to evaluate the effects of diesel exhaust particles (DEP) on the ocular surface, different concentrations (100 and 1000 μg/ml) of DEP eye drops were administered on the mouse ocular surface for a period of 28 days. After DEP treatment, the corneal epithelial permeability to Oregon Green Dextran was studied, which increased proportionally with time. Also, the number of corneal epithelial cell layers significantly increased, which was accompanied with a high Ki67 expression. On the other hand, the number of goblet cells in the conjunctival fornix were reduced, and apoptotic cells were detected in the corneal and conjunctival epithelium by TUNEL assay in the DEP treated group, along with increased Caspase 3/8 expression. Furthermore, the number of CD4 positive cells significantly increased in the conjunctiva, while NF-κB p65 (phospho S536) expression was elevated in the cornea and also the conjunctiva. Our data revealed that the topical administration of DEP on the ocular surface in mouse disrupted the organized structure of the ocular surface and induced an inflammation of the cornea and conjunctiva. [ABSTRACT FROM AUTHOR]

Objective: Fcγ receptor IIb (FcγRIIb) is an essential negative regulator of B cells that blocks B cell receptor (BCR) signaling and triggers c‐Abl–dependent apoptosis of B cells. FcγRIIb‐deficient mice display splenomegaly with expansion of B cells, leading to lupus. FcγRIIb‐I232T is a hypofunctional polymorphism associated with lupus susceptibility in humans, an autoimmune disease linked to diminished deletion of autoreactive B cells. In the context of the FcγRIIb‐I232T polymorphism, we investigated the role of FcγRIIb in the deletion of low‐affinity germinal center (GC) B cells, an important mechanism for preventing autoimmunity. Methods: We generated FcγRIIb232T/T mice to mimic human FcγRIIb‐I232T carriers and immunized mice with chicken gamma globulin (CGG)–conjugated NP, a T cell–dependent antigen, to examine the response of GC B cells. Results: Compared to wild‐type (WT) mice, FcγRIIb232T/T mice showed increased numbers of low‐affinity NP‐specific IgG and NP‐specific B cells and plasma cells; additionally, the expression of a somatic mutation (W33L) in their VH186.2 genes encoding high‐affinity BCR was reduced. Notably, FcγRIIb232T/T mice had a higher number of GC light zone B cells and showed less apoptosis than WT mice, despite having equivalent follicular helper T cell numbers and function. Moreover, phosphorylation of c‐Abl was reduced in FcγRIIb232T/T mice, and treatment of WT mice with the c‐Abl inhibitor nilotinib during the peak of GC response resulted in reduced affinity maturation reminiscent of FcγRIIb232T/T mice. Conclusion: Our findings provide evidence of a critical role of FcγRIIb/c‐Abl in the negative selection of GC B cells in FcγRIIb232T/T mice. Importantly, our findings indicate potential benefits of up‐regulating FcγRIIb expression in B cells for treatment of systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

The traditional Chinese medicine (TCM) You-Gui-Wan (YGW) has been used to treat asthma for hundreds of years. YGW is composed of 10 types of medicinal materials. However, the immune mechanism of YGW in asthma treatment has not been elucidated. Therefore, this study investigated asthma symptoms attenuated by YGW and the underlying immune regulatory mechanism. Intratracheal (i.t.) stimulation of BALB/c mice with Dermatophagoides pteronyssinus (Der p) was performed once per week (40 μL, 2.5 μg/μL). For six consecutive weeks, different doses of YGW (0.2 g/kg and 0.5 g/kg) were orally administered 30 min before stimulation with Der p. After the last stimulation, airway hyperreactivity, lung gene expression, and total immunoglobulin E (IgE) in blood were evaluated using a whole-body plethysmograph system, real-time PCR, and ELISA, respectively. In addition, DNP-IgE/DNP-BSA was added to stimulate mast cells (RBL-2H3), and YGW or various compound compositions (Trial) were added to RBL-2H3 cells for 30 min to evaluate the effects of the drug on mast cell degranulation and on gene expression. JMP 5.1 software was used to design and analyze YGW's critical compounds by which it inhibited ALOX-5 and HDC gene expression in RBL-2H3 cells. YGW significantly decreased serum total IgE levels and airway hyperresponsiveness in asthmatic mice. YGW also reduced the gene expression of IL-6, TNF-α, IL-4, IL-13, and COX-2 in the lungs of asthmatic mice and RBL-2H3 cells. YGW and the compound (Trial 21) present in YGW inhibited the gene expression of ALOX-5 and HDC in RBL-2H3 cells. The experimental results indicate that YGW exhibits anti-airway hyperresponsiveness and specific immunomodulatory effects. In addition, YGW synergistically inhibits ALOX-5 and HDC gene expression in mast cells through a combination of 21 compounds, including luteolin, quercetin, and β-carotene. [Display omitted] • You-Gui-Wan extract decreased Der p - induced airway hyperresponsiveness and total serum IgE levels in a mouse model of chronic asthma. • You-Gui -Wan extract inhibited the gene expression of IL-6, TNF-α, IL-4, IL-13 and COX-2 in the lungs of asthmatic mice and RBL-2H3 cells. • You-Gui -Wan extract and the compounds (Trail 21) contained in You-Gui -Wan inhibited the gene expression of ALOX-5 and HDC in RBL-2H3 cells. [ABSTRACT FROM AUTHOR]

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK. [ABSTRACT FROM AUTHOR]

Tanshinone IIA, one of the most pharmacologically bioactive phytochemicals isolated from Salvia miltiorrhiza Bunge, possesses several biological activities such as anti-inflammation, anti-cancer, neuroprotection and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effect of tanshinone IIA in hepatic cells. We demonstrated that tanshinone IIA significantly increased the amount of low-density lipoprotein receptor (LDLR) and LDL uptake activity in HepG2 cells at the post-transcriptional regulation. We further demonstrated that tanshinone IIA inhibited the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and mature protein, which may lead to an increase the cell-surface LDLR in hepatic cells. We further identified a regulatory DNA element involved in the tanshinone IIA-mediated PCSK9 down-regulation, which is located between the -411 and -336 positions of the PCSK9 promoter. Moreover, we found that tanshinone IIA markedly increased the nuclear forkhead box O3a (FoxO3a) level, enhanced FoxO3a/PCSK9 promoter complexes formation and decreased the PCSK9 promoter binding capacity of hepatocyte nuclear factor 1α (HNF-1α), resulting in suppression of PCSK9 gene expression. Finally, we found that the statin-induced PCSK9 overexpression was attenuated and the LDLR activity was elevated in a synergic manner by combination of tanshinone IIA treatment in HepG2 cells. Overall, our results reveal that the tanshinone IIA modulates LDLR level and activity via down-regulation of PCSK9 expression in hepatic cells. Our current findings provide a molecular basis of tanshinone IIA to develop PCSK9 inhibitors for cholesterol management. [ABSTRACT FROM AUTHOR]

GlnR-mediated repression of the GlnR regulon at acidic pH is required for optimal acid tolerance in Streptococcus mutans, the etiologic agent for dental caries. Unlike most streptococci, the GlnR regulon is also regulated by newly identified PmrA (SMUGS5_RS05810) at the transcriptional level in S. mutans GS5. Results from gel mobility shift assays confirmed that both GlnR and PmrA recognized the putative GlnR box in the promoter regions of the GlnR regulon genes. By using a chemostat culture system, we found that PmrA activated the expression of the GlnR regulon at pH 7, and that this activation was enhanced by excess glucose. Deletion of pmrA (strain ΔPmrA) reduced the survival rate of S. mutans GS5 at pH 3 moderately, whereas the GlnR mutant (strain ΔGlnR) exhibited an acid-sensitive phenotype in the acid killing experiments. Elevated biofilm formation in both ΔGlnR and ΔPmrA mutant strains is likely a result of indirect regulation of the GlnR regulon since GlnR and PmrA regulate the regulon differently. Taken together, it is suggested that activation of the GlnR regulon by PmrA at pH 7 ensures adequate biosynthesis of amino acid precursor, whereas repression by GlnR at acidic pH allows greater ATP generation for acid tolerance. The tight regulation of the GlnR regulon in response to pH provides an advantage for S. mutans to better survive in its primary niche, the oral cavity. [ABSTRACT FROM AUTHOR]

Background: Consumption of cocoa-derived polyphenols has been associated with several health benefits; however, their effects on the intestinal microbiome and related features of host intestinal health are not adequately understood.Objective: The objective of this study was to determine the effects of eating flavanol-enriched cocoa powder on the composition of the gut microbiota, tissue metabolite profiles, and intestinal immune status.Methods: Male pigs (5 mo old, 28 kg mean body weight) were supplemented with 0, 2.5, 10, or 20 g flavanol-enriched cocoa powder/d for 27 d. Metabolites in serum, urine, the proximal colon contents, liver, and adipose tissue; bacterial abundance in the intestinal contents and feces; and intestinal tissue gene expression of inflammatory markers and Toll-like receptors (TLRs) were then determined.Results: O-methyl-epicatechin-glucuronide conjugates dose-dependently increased (P< 0.01) in the urine (35- to 204-fold), serum (6- to 186-fold), and adipose tissue (34- to 1144-fold) of pigs fed cocoa powder. The concentration of 3-hydroxyphenylpropionic acid isomers in urine decreased as the dose of cocoa powder fed to pigs increased (75-85%,P< 0.05). Compared with the unsupplemented pigs, the abundance ofLactobacillusspecies was greater in the feces (7-fold,P= 0.005) and that ofBifidobacteriumspecies was greater in the proximal colon contents (9-fold,P= 0.01) in pigs fed only 20 or 10 g cocoa powder/d, respectively. Moreover, consumption of cocoa powder reducedTLR9gene expression in ileal Peyer's patches (67-80%,P< 0.05) and mesenteric lymph nodes (43-71%,P< 0.05) of pigs fed 2.5-20 g cocoa powder/d compared with pigs not supplemented with cocoa powder.Conclusion: This study demonstrates that consumption of cocoa powder by pigs can contribute to gut health by enhancing the abundance ofLactobacillusandBifidobacteriumspecies and modulating markers of localized intestinal immunity. [ABSTRACT FROM AUTHOR]

Background: Elafin inhibits serine proteases, such as human neutrophil elastase and proteinase 3, to prevent excessive damage during inflammation. However, the relationship between elafin and asthma is still unclear. Microarray technology was used to evaluate smoking- and asthma-related biomarkers in a discovery-driven manner. We identified candidate genes, e.g., proteinase inhibitor 3 (PI3), related to asthma and smoking from gene expression microarray data sets and evaluated their potential as biomarkers for asthma. Methods: We used human genome microarray data sets from smoking- and asthma-related gene expression data sets and performed real-time quantitative polymerase chain reaction to measure and validate differences in gene expression. We also recruited adult patients with asthma and age- and sex-matched control patients who were administered a structured questionnaire and evaluated for lung function and plasma elafin levels, which are encoded by the PI3 gene. Results: Six significantly altered candidate genes, PI3, protein kinase C iota, phosphoserine phosphatase, IQ motif-containing GTPase activating protein 1, interleukin 13 receptor α 1, and signal transducing adaptor molecule SH3 domain and ITAM motif 2, were identified from comparisons across the four asthma- and four smoking-related data sets included in this study. An in vitro study of human airway epithelial cells (A549) and a human monocytic cell line (THP-1) demonstrated that PI3 messenger RNA levels were significantly altered by nicotine exposure. Elafin concentration was significantly higher in control patients than in patients with asthma (p < 0.001). The plasma elafin concentration in the highest quartile (≥12.69 ng/mL) was inversely associated with asthma (adjusted odds ratio 0.122 [95% confidence interval, 0.053-0.278]) compared with the lowest quartile (<5.82 ng/mL) after adjusting for age, sex, smoking status, waist-to-hip ratio, percentage predicted forced expiratory volume in 1 second, cockroaches in the home, incense burning, and family history. Conclusion: Our study revealed that high elafin levels identified in smoking- and asthma-related microarray data sets and an epidemiologic study significantly reduced the risk of asthma. Further studies of elafin as a potential therapy for asthma are warranted. [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Facial motor neurons (FMNs) are involved in the remodeling of the facial nucleus in response to peripheral injury. This study aimed to examine the gene expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) in reinnervating dormant FMNs after facial nerve axotomy.Study Design: Animal study.Methods: Rat models of facial-facial anastomosis were set up and raised until the 90th day. By laser capture microdissection (LCM), the reinnervating neurons labeled by Fluoro-Ruby (FR) were first captured, and the remaining (dormant) neurons identified by Nissl staining were captured in the facial nucleus of the operated side. The total RNA of two types of neurons were extracted, and the gene expressions of AMPAR and NMDAR were studied by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).Results: Messenger RNA (mRNA) of AMPAR subunits (GluR1, GluR2, GluR3, and GluR4) and NMDAR subunits (NR1, NR2a, NR2b, NR2c, and NR2d) was detected in reinnervating and dormant neurons. The relative ratios exhibited that the expressions of GluR1, GluR4, NR2a, NR2b, NR2c, and NR2d mRNA were lower, whereas the expressions of GluR2, GluR3, and NR1 mRNA were higher in dormant FMNs than in reinnervating counterparts.Conclusions: LCM in combination with real-time qRT-PCR can be employed for the examination of gene expression of different FMNs in a heterogeneous nucleus. The adaptive changes in AMPAR and NMDAR subunit mRNA might dictate the regenerative fate of FMNs in response to the peripheral axotomy and thereby play a unique role in the pathogenesis of facial nerve injury and regeneration. [ABSTRACT FROM AUTHOR]

Although negative pressure wound therapy (NPWT) has been suggested for the management of acute and chronic wounds, the underlying mechanism of NPWT on the cellular level is not fully understood. Using a primary fibroblast culture model with controlled negative pressure, the cellular responses to the negative pressure was investigated. The present results showed that cell proliferation of fibroblasts was not affected under negative pressure. The mRNA expressions of collagen type I, collagen type III, and fibronectin were down-regulated when cells treated with negative pressure for once only. However, those mRNA expressions were up-regulated when cells subjected to periodical negative pressure consecutively for 5 days. The protein production of TGF-β1 was enhanced, while the TIMP-1, decorin, and IL-6 were not influenced. The pro-MMP-9 was not stimulated when fibroblasts subjected to negative pressure. The regulations of extracellular matrix production in fibroblasts provides an insight into the cellular mechanisms of NPWT. [ABSTRACT FROM AUTHOR]

Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs. [ABSTRACT FROM AUTHOR]

Protein ubiquitinylation regulates protein stability and activity. RAD6, an E2 ubiquitin-conjugating enzyme, which that has been substantially biochemically characterized, functions in a number of biologically relevant pathways, including cell cycle progression. In this study, we show that RAD6 promotes the G1-S transition and cell proliferation by regulating the expression of cyclin D1 (CCND1) in human cells. Furthermore, our data indicate that RAD6 influences the transcription of CCND1 by increasing monoubiquitinylation of histone H2B and trimethylation of H3K4 in the CCND1 promoter region. Our study presents, for the first time, an evidence for the function of RAD6 in cell cycle progression and cell proliferation in human cells, raising the possibility that RAD6 could be a new target for molecular diagnosis and prognosis in cancer therapeutics. [ABSTRACT FROM AUTHOR]

Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress. [ABSTRACT FROM AUTHOR]

Retinal post-mitotic neurocytes display genomic instability after damage induced by physiological or pathological factors. The involvement of BRCA1, an important factor in development and DNA repair in mature retinal neurocytes remains unclear. Thus, we investigated the developmental expression profile of BRCA1 in the retina and defined the role of BRCA1 in DNA repair in retinal neurocytes. Our data show the expression of BRCA1 is developmentally down-regulated in the retinas of mice after birth. Similarly, BRCA1 is down-regulated after differentiation induced by TSA in retinal precursor cells. An end-joining activity assay and DNA fragmentation analysis indicated that the DNA repair capacity is significantly reduced. Moreover, DNA damage in differentiated cells or cells in which BRCA1 is silenced by siRNA interference is more extensive than that in precursor cells subjected to ionizing radiation. To further investigate non-homologous end joining (NHEJ), the major repair pathway in non-divided neurons, we utilized an NHEJ substrate (pEPI-NHEJ) in which double strand breaks are generated by I-SceI. Our data showed that differentiation and the down-regulation of BRCA1 respectively result in a 2.39-fold and 1.68-fold reduction in the total NHEJ frequency compared with that in cells with normal BRCA1. Furthermore, the analysis of NHEJ repair junctions of the plasmid substrate indicated that BRCA1 is involved in the fidelity of NHEJ. In addition, as expected, the down-regulation of BRCA1 significantly inhibits the viability of retina precursor cells. Therefore, our data suggest that BRCA1 plays a critical role in retinal development and repairs DNA damage of mature retina neurocytes. [ABSTRACT FROM AUTHOR]

Background: Tetramethylpyrazine (TMP) is one of the active ingredients extracted from the Chinese herb Chuanxiong, which has been used to treat cerebrovascular and cardiovascular diseases, pulmonary diseases and cancer. However, the molecular mechanisms underlying the actions of TMP have not been fully elucidated. In a previous study we showed that TMP-mediated glioma suppression and neural protection involves the inhibition of CXCR4 expression. The SDF-1/CXCR4 axis plays a fundamental role in many physiological and pathological processes. In this study, we further investigated whether the regulation of the SDF-1/CXCR4 pathway is also involved in the TMP-mediated inhibition of neovascularization or fibrosis and improvement of microcirculation. Methodology/Principal Findings: Using a scratch-wound assay, we demonstrated that TMP significantly suppressed the migration and tubule formation of the human umbilical vein endothelial cell line ECV304 in vitro. The expression of CXCR4 in ECV304 cells is notably down-regulated after TMP treatment. In addition, TMP significantly suppresses corneal neovascularization in a rat model of corneal alkali burn injury. The expression of CXCR4 on days 1, 3 and 7 post-injury was determined through RT-PCR analysis. Consistent with our hypotheses, the expression of CXCR4 in the rat cornea is significantly increased with alkali burn and dramatically down-regulated with TMP treatment. Moreover, TMP treatment significantly attenuates bleomycin-induced rat pulmonary fibrosis, while immunofluorescence shows a notably decreased amount of CXCR4-positive cells in the TMP-treated group. Furthermore, TMP significantly down-regulates the expression of CXCR4 in platelets, lymphocytes and red blood cells. Whole-blood viscosity and platelet aggregation in rats are significantly decreased by TMP treatment. Conclusions: These results show that TMP exerts potent effects in inhibiting neovascularization, fibrosis and thrombosis under pathological conditions; thus, the underlying mechanism of TMP might partially contribute to the down-regulation of CXCR4. [ABSTRACT FROM AUTHOR]

RBR (RING1-IBR-RING2) proteins play an important role in protein ubiquitination and are involved in many cellular processes. Recent studies showed plant RBR genes were induced by abiotic and biotic stresses. However, detailed studies on RBR genes in the important oil crop, soybean (Glycine max (L.) Merr.), is still lacking. Here we performed a genome-wide search and identified 24 RBR domain-containing genes from the soybean genome sequence and cloned 11 of them. Most soybean RBR proteins contain a highly conserved RBR supra-domain. Phylogenetic analyses indicated all 24 soybean RBR proteins are most related to the RBR proteins from Phaseolus vulgaris, and could be classified into seven groups including Ariadne A, Ariadne B, ARA54, Plant IIA, Plant IIB, Plant IIC, and Helicase. Tandem duplication and block duplication were found among the Ariadne B and Plant IIC group of soybean RBR genes. Despite the conserved RBR supra-domain, there are extensive variations in the additional protein motifs and exon-intron structures between different groups, which indicate they might have diverse functions. Most soybean RBR proteins are predicted to localize in nucleus, and four of them were experimentally confirmed by GFP fusion proteins. Soybean RBR genes are broadly expressed in many tissue types with a little more abundant in the roots and flowers than leaves, stems, and seeds. The expression of GmRTRTP3 (Plant IIB) and GmRTRTP5 (Plant IIC) are induced by NaCl treatment, which suggests these RBR genes might be involved in soybean response to abiotic stresses. [ABSTRACT FROM AUTHOR]