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Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners.

There exists an inverse relationship between the rate of molecular evolution and the level of gene expression. Among the many explanations, the "toxic-error" hypothesis is a most general one, which posits that processing errors may often be toxic to the cells. However, toxic errors that constrain the evolution of highly expressed genes are often difficult to measure. In this study, we test the toxic-error hypothesis by using microRNA (miRNA) genes because their processing errors can be directly measured by deep sequencing. A miRNA gene consists of a small mature product (≈22 nt long) and a "backbone." Our analysis shows that (i) like the mature miRNA, the backbone is highly conserved; (ii) the rate of sequence evolution in the backbone is negatively correlated with expression; and (iii) although conserved between distantly related species, the error rate in miRNA processing is also negatively correlated with the expression level. The observations suggest that, as a miRNA gene becomes more highly (or more ubiquitously) expressed, its sequence evolves toward a structure that minimizes processing errors.

Purpose: Optineurin is a pathogenic gene associated with primary open angle glaucoma (POAG), in which the retinal ganglion cells (RGCs) are targeted. However, the functions of optineurin, particularly in RGCs, are currently not clear. We introduced optineurin siRNA into cultured retinal ganglion cell 5 (RGC-5) and PC12 cells to determine the cellular and molecular mechanisms underlying the role of optineurin in POAG., Methods: We constructed four optineurin siRNA-expressing plasmids, and transiently transfected them into PC12 cells. Quantitative real-time PCR, western blot, and fluorescent microscopy were used to determine optineurin expression and select the most effective optineurin siRNA to construct RGC-5 and PC12 stable transfected cells. Dimethylthiazolyl diphenyl tetrazolium bromide (MTT) assay and flow cytometry were applied to investigate the role of optineurin siRNA in cell growth and apoptosis. Gene microarray and quantitative real-time PCR were used to screen and validate differentially expressed genes in optineurin siRNA transfected PC12 and RGC-5 cells., Results: siRNA effectively downregulated optineurin expression in RGC-5 and PC12 stable transfected cells. Optineurin siRNA significantly inhibited cell growth and increased apoptosis in RGC-5 and PC12 cells. Microarray analysis identified 112 differentially expressed genes in optineurin siRNA transfected RGC-5 cells. Quantitative real-time PCR and western blot confirmed that the expression of brain-derived neurotrophic factor (Bdnf), neurotrophin-3(Ntf3), synaptosomal-associated protein 25(Snap25), and neurofilament, light polypeptide(Nefl) was significantly downregulated in RGC-5 and PC12 cells transfected with optineurin siRNA., Conclusions: Our study suggested that optineurin downregulation by siRNA in RGCs was an in vitro model for studying the mechanisms of optineurin effects on POAG. Neuroprotective factor and axonal transport genes may be involved in the development of POAG and could be novel targets for treating POAG due to optineurin mutation.

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Motivation A growing amount of noncoding genetic variants, including single-nucleotide polymorphisms, are found to be associated with complex human traits and diseases. Their mechanistic interpretation is relatively limited and can use the help from computational prediction of their effects on epigenetic profiles. However, current models often focus on local, 1D genome sequence determinants and disregard global, 3D chromatin structure that critically affects epigenetic events. Results We find that noncoding variants of unexpected high similarity in epigenetic profiles, with regards to their relatively low similarity in local sequences, can be largely attributed to their proximity in chromatin structure. Accordingly, we have developed a multimodal deep learning scheme that incorporates both data of 1D genome sequence and 3D chromatin structure for predicting noncoding variant effects. Specifically, we have integrated convolutional and recurrent neural networks for sequence embedding and graph neural networks for structure embedding despite the resolution gap between the two types of data, while utilizing recent DNA language models. Numerical results show that our models outperform competing sequence-only models in predicting epigenetic profiles and their use of long-range interactions complement sequence-only models in extracting regulatory motifs. They prove to be excellent predictors for noncoding variant effects in gene expression and pathogenicity, whether in unsupervised "zero-shot" learning or supervised "few-shot" learning. Availability and implementation Codes and data can be accessed at https://github.com/Shen-Lab/ncVarPred-1D3D and https://zenodo.org/record/7975777. [ABSTRACT FROM AUTHOR]

Background: Genetic mutations of IKZF1 have been frequently delineated in B‐lineage acute leukaemia (B‐ALL) but rarely elucidated in acute myeloid leukaemia (AML). IKZF1 mutations confer a poor prognosis in AML, and hotspot mutations of IKZF1, N159Y and N159S tend to occur in B‐ALL and AML respectively. However, the pathogenesis of IKZF1 N159S in AML and IKZF1 lineage susceptibility are largely unknown. Methods: The genetic and clinical characteristics of IKZF1‐mutated AML patients were evaluated. Multi‐omics analysis and functional assays were performed in vitro using IKZF1 mutations knock‐in AML cell lines. Results: 23 (4.84%) small sequence variants of IKZF1 were identified in 475 newly diagnosed AML (non‐M3) patients. Based on RNA sequencing, three classes of IKZF1‐related AML were defined, including 9 patients (39.13%) with IKZF1 N159S mutations, 10 (43.47%) with CEBPA mutations and 4 others (17.39%). IKZF1 N159S may define a unique subgroup with higher HOXA/B expression and native B‐cell immune fractions. Gene expression data of multiple knock‐in cell lines indicate that the lymphocyte differentiation‐related MME and CD44 kept high expression in IKZF1 N159Y but were downregulated in N159S. CUT&TAG sequencing showed that IKZF1 N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis of IKZF1 N159S may depend on the deregulation of several cofactors, such as oncogenic MYC and CPNE7 targets. Conclusions: Collectively, we dissected the molecular spectrum and clinical features of IKZF1‐related AML, which may promote an in‐depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML. [ABSTRACT FROM AUTHOR]

Background: Bioinformatics showed that long non-coding RNA (lncRNA) pgm5-as1 was regulated in patients with colorectal cancer (CRC), and miR-484 was also regulated in CRC. We aimed at determining the modulatory pathway of lncRNA pgm5-as1 in CRC cells, and whether miR-484 was involved in the pathway. Materials and Methods: The target gene of pgm5-as1 was predicted by bioinformatics and verified by dual luciferase assay. Transcription levels of pgm5-as1 and miR-484 were determined by quantitative real-time polymerase chain reaction. Viability, migration rate, invasion, and growth of SW480 and HCT116 cells were determined by Cell Counting Kit-8 (CCK-8), wound healing assay, transwell, and colony formation assay, respectively. Results:pgm5-as1 was upregulated in CRC tissues and cell lines; however, its downregulation contributed to the decreasing of cell viability, growth, migration, and invasion of SW480 and HCT116 cells. Moreover, miR-484 was predicted as the target of pgm5-as1, and the downregulation of pgm5-as1 partially restored the elevated cell viability, growth, migration, and invasion that were induced by the inhibition of miR-484 expression in SW480 and HCT116 cells. Conclusions: The loss of miR-484 expression in CRC might be involved in the promotion and metastasis of CRC, which may be caused by the overexpression of pgm5-as1. Hence, the downregulation of pgm5-as1 could be a therapeutic target in the prevention or intervention of CRC. [ABSTRACT FROM AUTHOR]

Thrombin plays a pivotal role in the pathogenesis of atherosclerosis. Baicalin, an active flavonoid compound, was shown to attenuate the development of atherosclerosis, but the mechanism remains elusive. In the present study, the role and mechanism of baicalin in thrombin-induced cell injury was investigated in human umbilical vein endothelial cells (HUVECs). Our results showed that baicalin significantly reduced thrombin-induced apoptosis of HUVECs. Additional experiments showed that baicalin inhibited thrombin-induced NF-κB activation and PAR-1 expression. In addition, baicalin decreased thrombin-induced PAR-1 expression by inhibiting ERK pathway. These results indicated that baicalin has protective effects on thrombin-induced cell injury in HUVECs possibly through inhibition of PAR-1 expression and its downstream NF-κB activation, which was mediated by ERK1/2 activation. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LAD) is difficult to diagnose as it tends to be small in size and metastasize early. The aim of the present study was to investigate prognostic factors for patients with LAD and establish a prognosis risk model. A training set consisting of clinical and RNA sequencing data from 503 patients with LAD, as well as expression data from a further 59 LAD and adjacent tissues, was obtained from The Cancer Genome Atlas. Additionally, a validation dataset was acquired from the Gene Expression Omnibus database (GSE26939), which included clinical and gene expression data from 115 patients. Using the DESeq2 package to compare expression between LAD and adjacent tissues, differentially expressed genes (DEGs) were identified. On the basis of survival and the random forests for survival, regression and classification package, genes for constructing the prognosis risk model were selected. The prognosis risk model was constructed and validated using the survival package. Subsequently, high- and low-risk groups were compared using the Limma package to identify DEGs, and enrichment analysis was performed using the web-based gene set analysis toolkit. A protein-protein interaction network was visualized using Cytoscape software. There were 18,567 DEGs between the LAD samples and the adjacent tissues, and 363 DEGs between the high- and low-risk groups. Of these, four genes were selected for constructing the prognosis risk model, myosin IE (MYO1E), endoplasmic reticulum oxidoreductase 1α (ERO1L), C1q and tumor necrosis factor-related protein 6 (C1QTNF6) and family with sequence similarity 83, member A (FAM83A). The survival time of high- and low-risk groups in the validation set were significantly different. Functional enrichment revealed that the genes that interacted with MYO1E, ERO1L, C1QTNF6 and FAM83A separately were enriched in 'cell cycle regulation', 'synthesis and assembly of nucleic acids', 'histone modification and cell cycle progression' and 'cell secretion process'. The four-gene prognosis risk model could potentially be used for predicting the survival of patients with LAD. [ABSTRACT FROM AUTHOR]

Choline kinase a (CHKA), the enzyme that converts choline to phosphocholine, has been studied in human carcinogenesis widely. However, the expression and underlying clinicopathological characteristics of CHKA in lung adenocarcinoma remains elusive. In the present study, a tissue microarray of 119 pairs of lung adenocarcinoma samples and corresponding adjacent normal mucosae was used to analysis CHKA expression by immunohistochemistry, and CHKA was observed to exhibit enhanced expression in lung adenocarcinoma tissues. Elevated CHKA expression in lung adenocarcinoma tissues at the gene and protein level was observed. The levels of CHKA expression were closely associated with the poor prognosis status of lung adenocarcinoma patients. Furthermore, certain clinicopathological characteristics such as tumor diameter and differentiation were observed to be significant in those lung adenocarcinoma patients who displayed enhanced CHKA expression. The analysis of CHKA expression could provide a more precise way to predict the prognosis of lung adenocarcinoma patients. Collectively, the present study revealed a novel biomarker in lung adenocarcinoma, and indicated that CHKA may be a promising prognostic marker and therapeutic target for lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is a well-known estrogenic endocrine disrupting chemical (EDC) ubiquitously present in various environmental media. The present study aims to identify the responsive genes in male fish chronically exposed to low concentrations of BPA at the transcription level. We screened genes from a suppression subtractive hybridization library constructed from male medaka ( Oryzias latipes ) livers after 60-d exposure to 10 μg/L BPA under the condition at which changes of hepatic antioxidant parameters have been previously reported. The identified genes were predicted to be involved in multiple biological processes including antioxidant physiology, endocrine system, detoxification, notably associated with the immune response processes. With real time PCR analysis, the immune-associated genes including hepcidin-like precursor , complement component and factors , MHC class I , alpha-2-macroglobulin and novel immune-type receptor 6 isoform were significantly up-regulated in a nonmonotonic dose response pattern in livers upon exposure to different concentrations of BPA (0.1, 1, 10, 100, 1000 μg/L). Our results demonstrated a negative impact on gene regulation in fish chronically exposed to relatively low and environmentally relevant concentrations of BPA, and suggested the potential immune modulatory effect of chronic EDC exposure on fish. The immunotoxicity of BPA and other EDCs should be much concerned for the health of human beings and other vertebrates exposed to it. [ABSTRACT FROM AUTHOR]

The incidence rate of human uterine leiomyomas is over 70% in the women of childbearing age, which has caused serious health and financial burden. Our previous study confirmed that Bisphenol A (BPA)，representative environmental estrogen, promoted the proliferation of human uterine leiomyomas and up-regulated the expression of cell proliferation-related genes. In this study, by combining ChIP-seq and RNA-seq, it was shown that after BPA intervention, H3K27ac modification levels and gene expression levels were altered in uterine leiomyomas cells. Moreover experimental verification found that BPA can regulate ITGA2 through the transcription factor XBP1, activate the downstream PI3K/AKT signaling pathway, eventually promote the proliferation of uterine leiomyomas. The present study provides new insights into the pathogenesis associated with exposure to BPA and other endocrine disruptors with similar effects by defining XBP1 as an important regulator, and which may act as an intervention and treatment target for uterine leiomyomas. [Display omitted] • BPA promotes the proliferation of uterine leiomyomas in vivo and vitro. • Integrated RNA-seq and ChIP-seq analysis to study the effect of BPA. • XBP1 as a regulator in Bisphenol A induced uterine leiomyoma cell proliferation. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common chronic and progressive neurodegenerative disorder. Its etiology remains elusive and at present only symptomatic treatments exists. Helicobacter pylori chronically colonizes the gastric mucosa of more than half of the global human population. Interestingly, H. pylori positivity has been found to be associated with greater of PD motor severity. In order to investigate the underlying cause of this association, the Sengenics Immunome protein array, which enables simultaneous screening for autoantibodies against 1636 human proteins, was used to screen the serum of 30 H. pylori-seropositive PD patients (case) and 30 age- and gender-matched H. pylori-seronegative PD patients (control) in this study. In total, 13 significant autoantibodies were identified and ranked, with 8 up-regulated and 5 down-regulated in the case group. Among autoantibodies found to be elevated in H. pylori-seropositive PD were included antibodies that recognize Nuclear factor I subtype A (NFIA), Platelet-derived growth factor B (PDGFB) and Eukaryotic translation initiation factor 4A3 (eIFA3). The presence of elevated autoantibodies against proteins essential for normal neurological functions suggest that immunomodulatory properties of H. pylori may explain the association between H. pylori positivity and greater PD motor severity. [ABSTRACT FROM AUTHOR]

Background: To examine the circulating microRNA (miRNA) expression profile in a mouse model of diet-induced obesity (DIO) with subsequent weight reduction achieved via low-fat diet (LFD) feeding. Results: Eighteen C57BL/6NCrl male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal %) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal % high-fat diet (HFD) for 12 weeks; and (3) DIO + LFD, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal % LFD for 4 weeks. A switch to LFD feeding led to decreases in body weight, adiposity, and blood glucose levels in DIO mice. Microarray analysis of miRNA using The Mouse & Rat miRNA OneArray® v4 system revealed significant alterations in the expression of miRNAs in DIO and DIO + LFD mice. Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice. Target prediction and function annotation of associated genes revealed that these genes were predominantly involved in metabolic, insulin signaling, and adipocytokine signaling pathways that directly link the pathophysiological changes associated with obesity and weight reduction. Conclusions: These results imply that obesity-related reductions in the expression of circulating miRNAs could be reversed through changes in metabolism associated with weight reduction achieved through LFD feeding. [ABSTRACT FROM AUTHOR]

The migration of vascular endothelial cells (ECs) is essential for reendothelialization after implantation of cardiovascular biomaterials. Reendothelialization is largely determined by surface properties of implants. In this study, surfaces modified with various chemical functional groups (CH 3 , NH 2 , COOH, OH) prepared by self-assembled monolayers (SAMs) were used as model system. Expressions and distributions of critical proteins in the integrin-induced signaling pathway were examined to explore the mechanisms of surface chemistry regulating EC migration. The results showed that SAMs modulated cell migration were in the order CH 3 > NH 2 > OH > COOH, determined by differences in the expressions of focal adhesion components and Rho GTPases. Multiple integrin subunits showed difference in a surface chemistry-dependent manner, which induced a stepwise activation of signaling cascades associated with EC migration. This work provides a broad overview of surface chemistry regulated endothelial cell migration and establishes association among the surface chemistry, cell migration behavior and associated integrin signaling events. Understanding the relationship between these factors will help us to understand the surface/interface behavior between biomaterials and cells, reveal molecular mechanism of cells sensing surface characterization, and guide surface modification of cardiovascular implanted materials. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) patients exhibit sleep and circadian disturbances prior to the onset of cognitive decline, and these disruptions worsen with disease severity. However, the molecular mechanisms behind sleep and circadian disruptions in AD patients are poorly understood. In this study, we investigated sleep pattern and circadian rhythms in Presenilin-1/2 conditional knockout (DKO) mice. Assessment of EEG and EMG recordings showed that DKO mice displayed increased NREM sleep time but not REM sleep during the dark phase compared to WT mice at the age of two months; at the age of six months, the DKO mice showed increased wakefulness periods and decreased total time spent in both NREM and REM sleep. WT exhibited time-of-day dependent modulation of contextual and cued memory. Compared with WT mice, 4-month-old DKO mice exhibited the deficiency regardless trained and tested in the same light/night phase or not. Particularly interesting was that DKO showed circadian modulation deficiency when trained in the resting period but not in the active period. Long noncoding RNAs (lncRNAs) are typically defined as transcripts longer than 200 nucleotides, and they have rhythmic expression in mammals. To date no study has investigated rhythmic lncRNA expression in Alzheimer's disease. We applied RNA-seq technology to profile hippocampus expression of lncRNAs in DKO mice during the light (/resting) and dark (/active) phases and performed gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of the cis lncRNA targets. Expression alteration of lncRNAs associated with immune response and metallodipeptidase activity may contribute to the circadian disruptions of DKO mice. Especially we identified some LncRNAs which expression change oppositely between day and light in DKO mice compared to WT mice and are worthy to be studied further. Our results exhibited the circadian rhythm sleep disorders and a noteworthy time-of-day-dependent memory deficiency in AD model mice and provide a useful resource for studying the expression and function of lncRNAs during circadian disruptions in Alzheimer's disease. • Loss of presenilin function altered sleep-wake pattern and circadian rhythms of mice. • Loss of presenilin function impaired time-of-day-dependent fear memory in mice. • lncRNAs associated with immune response and metallodipeptidase activity expression alteration in presenilin knockout mice. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To study the effect and mechanism of action of nonylphenol (NP), an environmental oestrogen, on uterine leiomyoma (UL) cells. Methods: Primary culture and subculture of human UL cells, identified as smooth muscle cells by immunocytochemical staining with a monoclonal anti-α-smooth muscle actin antibody, were performed. The viability of cells treated with various concentrations of NP for 24, 48 and 72h was determined by CCK-8 assay. mRNA expression of oestrogen receptor α (ERα), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was detected using real-time quantitative polymerase chain reaction, and protein expression was detected using Western blot analysis for all groups. Results: NP promoted the growth of UL cells and expression of ERα, IGF-1 and VEGF; this was positively correlated with the concentration and duration of NP treatment. Conclusion: NP promotes the growth of UL cells. The mechanism of action appears to be over-expression of IGF-1 and VEGF, up-regulated by ERα, resulting in the growth of UL cells. [Copyright &y& Elsevier]

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes. [ABSTRACT FROM AUTHOR]

ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene ( ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract: Secreted protein acidic and rich in cysteine (SPARC) is a secretory multifunctional matricellular glycoprotein. High circulating levels of SPARC have been reported to be associated with obesity and insulin resistance. The aim of the present study was to investigate whether SPARC induces insulin resistance and mitochondrial dysfunction in adipocytes. Our results showed that feeding high fat diet to rats for 12 weeks significantly increased SPARC expression in adipose tissues at both mRNA and protein levels. Moreover, SPARC overexpression in stably transfected 3T3-L1 cells induced insulin resistance and mitochondrial dysfunction, as evidenced by inhibition of insulin-stimulated glucose transport, lower ATP synthesis and mitochondrial membrane potential, reduced expression of glucose transporter 4 (GLUT4), and increased levels of reactive oxygen species (ROS) in mature adipocytes. Finally, overexpression of SPARC also modulated the expression levels of several inflammatory cytokines, which play important roles in insulin resistance, glucose and lipid metabolism during adipogenesis. In conclusion, our data suggest that SPARC is involved in obesity-induced adipose insulin resistance and may serve as a potential target in the treatment of obesity and obesity-related insulin resistance. [Copyright &y& Elsevier]

Abstract: Orexin A regulates food intake, energy metabolism and gastrointestinal function; it also increases glucose uptake and inhibits lipolysis, suggesting a role for orexin A in glucose and lipid metabolism. In this study, the effects of orexin A on glucose transporter 4 (GLUT4) mRNA level and lipid content were explored in 3T3-L1 preadipocytes and adipocytes. Orexin receptor 1 (OX1R) protein expression was determined in the adipose tissue of normal and obese rats. In addition, 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes were incubated with different concentrations of orexin A (10−9 to 10−7 M), without or with OX1R specific antagonist, then the peroxisome proliferator-activated receptor-γ2 (PPARγ2) mRNA expression was analyzed. Differentiated 3T3-L1 adipocytes were exposed to orexin A, without or with MAPK and OX1R antagonist, after which the GLUT4 and ERK1/2, JNK, and p38 MAPK activation, and triglyceride (TG) content were measured. We observed that OX1R protein expression was decreased in obese rats, and OX1R protein level was negatively correlated with body fat, Lee's index, TG, total cholesterol, and fasting insulin levels. Orexin A enhanced PPARγ2 mRNA expression in a dose-dependent manner in 3T3-L1 preadipocytes through OX1R. In differentiated 3T3-L1 adipocytes, orexin A significantly increased GLUT4 mRNA levels, which was blocked by the ERK1/2, JNK, and p38 MAPK inhibitors as well as OX1R antagonist. Furthermore, orexin A increased cellular TG content via ERK1/2, JNK, and p38 MAPK as well as OX1R. Thus, orexin A increases GLUT4 mRNA expression and lipid accumulation in differentiated 3T3-L1 adipocytes via ERK1/2, JNK, and p38 MAPK signaling. In addition, orexin A increases PPARγ2 mRNA expression in 3T3-L1 preadipocytes. Further studies are necessary to elucidate the impact of orexin A in metabolic disorders and adipocyte differentiation. [Copyright &y& Elsevier]

Yanggan Huayu granule (YGHY) is a formula of traditional Chinese medicine that has been widely used to treat patients with liver cancer. But its working mechanism is still poorly understood. Aim of the study: To investigate the anti-tumor effect of YGHY and its working mechanisms in hepatocellular carcinoma (HCC). H22 mouse xenograft model was used to detect the effect of YGHY on hepatocellular carcinoma (HCC). MTT and CCK8 assays were performed to assess the effect of YGHY on HCC cell growth. Transwell assay was performed to detect the invasion and migration activities of HCC cells. Effect of YGHY drug-contained serum on apoptosis was detected by flow cytometry. Western blot was performed to detect the protein expressions. Results showed that YGHY inhibited tumor volume and weight, induced the apoptosis of HepG2 and SMMC-7721 cells and increased the protein expressions of Cleaved-Caspase3 and Cleaved-PARP. Furthermore, YGHY significantly down-regulated the protein expression of p-AKT. SC79, as an activator of AKT signaling, was able to increase the expression of p-AKT, and regulate the protein expressions of Cleaved-Caspase3, Cleaved-PARP, BCL-2 and BAX. YGHY drug-contained serum negated the protein expression change provided by SC79. Taken together, this data indicates that YGHY could inhibit HCC growth by inducing apoptosis, operating through AKT signaling. [Display omitted] • YGHY inhibited hepatocellular carcinoma both in vivo and in vitro. • YGHY induced apoptosis of hepatocellular carcinoma cells. • YGHY induced apoptotic signaling mediated by Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Previously, we had identified a specific whole blood-derived microRNAs (miRNAs) signature in mice following in vivo injection of lipopolysaccharide (LPS) originated from Gram-negative bacteria. This study was designed to profile the circulating miRNAs expression in mice exposed to lipoteichoic acid (LTA) which is a major component of the wall of Gram-positive bacteria. Results: C57BL/6 mice received intraperitoneal injections of 100 μg of LTA originated from Bacillus subtilis, Streptococcus faecalis, and Staphylococcus aureus were killed 6 h and the whole blood samples were obtained for miRNA expression analysis using a miRNA array (Phalanx miRNA OneArrayw 1.0). Up-regulated expression of miRNA targets in the whole blood, serum and white blood cells (WBCs) of C57BL/6 and Tlr2-/- mice upon LTA treatment in 10, 100, or 1000 ug concentrations was quantified at indicated time (2, 6, 24, and 72 h) using real-time RT-PCR and compared with that in the serum of C57BL/6 mice injected with 100 ug of LPS. A significant increase of 4 miRNAs (miR-451, miR-668, miR-1902, and miR-1904) was observed in the whole blood and the serum in a dose- and time-dependent fashion following LTA injection. Induction of miRNA occurred in the serum after 2 h and persisted for at least 6 h. No increased expression of these 4 miRNAs was found in the WBCs. Higher but not significant expression level of these 4 miRNAs were observed following LTA treatment in the serum of Tlr2-/-against that of C57BL6 mice. In contrast, LPS exposure induced moderate expression of miR-451 but not of the other 3 miRNA targets. Conclusions: We identified a specific circulating miRNA signature in mice exposed to LTA. That expression profile is different from those of mice exposed to LPS. Those circulating miRNAs induced by LTA or LPS treatment may serve as promising biomarkers for the differentiation between exposures to Gram-positive or Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Summary: gp78 is a membrane-anchored ubiquitin ligase mediating the degradation of HMG-CoA reductase (HMGCR) and Insig-1. As a rate-limiting enzyme in cholesterol biosynthesis, HMGCR undergoes rapid sterol-promoted degradation. In contrast, destruction of Insig-1 releases its inhibition on SREBP and stimulates the expression of lipogenic genes. Thus, gp78 has opposite effects on lipid biosynthesis. We here generated liver-specific gp78 knockout (L-gp78 −/− ) mice and showed that although the degradation of HMGCR was blunted, SREBP was suppressed due to the elevation of Insig-1/-2, and therefore the lipid biosynthesis was decreased. The L-gp78 −/− mice were protected from diet-/age-induced obesity and glucose intolerance. The livers of L-gp78 −/− mice produced more FGF21, which activated thermogenesis in brown adipocytes and enhanced energy expenditure. Together, the major function of gp78 in liver is regulating lipid biosynthesis through SREBP pathway. Ablation of gp78 decreases the lipid levels and increases FGF21, and is beneficial to patients with metabolic diseases. [ABSTRACT FROM AUTHOR]

Abstract: NS1 protein of influenza A virus is involved in regulating the apoptosis of infected cells. We found that exogenously expressed NS1 was able to associate with the tumor suppressor p53 that plays an essential role in regulating apoptosis of influenza A virus-infected cells. Exogenous expression of NS1 resulted in inhibition of p53-mediated transcriptional activity and apoptosis. The p53 inhibitory domain of NS1 was located between amino acids 144 and 188. This domain is necessary for NS1 to inhibit p53 activity, but it requires additional region(s) to cooperatively exert this inhibitory function. [Copyright &y& Elsevier]

Summary: Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor- and Toll-like receptor-induced activation of nuclear factor-κB (NF-κB). A novel isoform of chicken MyD88, designated chicken MyD88-2, has been cloned and functionally characterized. Its open reading frame is of length 900bp, and it encodes a predicted 299 residue protein, similar in length to its mammalian orthologues, but, respectively, 77 and 69 amino acids shorter than the previously described chicken MyD88-1 and -3. The amino acid sequence of chicken MyD88-2 displays 96.9%, 96.9%, 70.4% and 70.2% identity with, respectively, chicken MyD88-1, -3, human and mouse MyD88. Chicken MyD88-2 expression was detected in a range of tissues tested, but no expression of either chicken MyD88-1 or -3 was observed. The over-expression of chicken MyD88-2 significantly induced the activation of NF-κB in vitro, suggesting that chicken MyD88-2 plays an important role in the innate immune responses of chicken. [Copyright &y& Elsevier]

The study aimed to recognize potential molecular targets and signal pathways whereby phenolic environmental estrogen promotes the proliferation of uterine leiomyoma cells. Primary cultured cell lines of uterine leiomyoma were treated with 0.1% DMSO, 10.0 μmol/L Bisphenol A (BPA), and 32.0 μmol/L Nonylphenol (NP) for 48 h before RNA-seq was performed. Those genes affected by BPA and NP were identified. Then, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Protein-protein Interaction (PPI) analysis were performed. Quantitative real-time polymerase chain reaction (q-PCR) and western blot were used to verify the differentially expressed gene and protein. Compared to with the control group, 739 differentially expressed genes were identified in both the BPA group and the NP group. GO enrichment analysis showed that the most enriched GO terms were connective tissue development and G1/S transition of mitotic cell cycle, and extracellular matrix. The results of KEGG enrichment analysis showed that differentially expressed mRNA were enriched mainly in three primary pathways, including environmental information processing, human diseases, and cellular processes. The cell cycle, PI3K-Akt signaling pathway are significantly enriched. The q-PCR and western blot verified the cell cycle associated genes and proteins were upregulated in both BPA group and NP group. Both BPA and NP activated the PI3K-AKT signaling pathway. Phenolic environmental estrogens may promote the proliferation and cell cycle progression of uterine leiomyoma cells through rapid non-genomic ER signaling, which leads to disordered cell cycle regulation and accelerates the transition of the cell cycle from G0/G1 phase to S phase. In addition, as an external stimulant, phenolic estrogen promotes the upregulation of inflammatory factors in uterine leiomyomas. • Phenolic environmental estrogens (EEs) promote uterine leiomyoma cell proliferation. • Phenolic EEs accelerates the transition of the cell cycle from G0/G1 to S phase. • RNA-seq was used to find out the genes regulated by BPA and NP together. • CCND1 and CCNE2 are the potential core molecular targets for phenolic EEs. • Phenolic EEs affect uterine leiomyomas by cell cycle and PI3K-Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: The rabies virus (RV) glycoprotein (G protein) induces neutralizing antibodies, which are important in protection against rabies. In the present study, three gene fragments that encode polypeptides (corresponding to amino acid residues 19–60, 181–219, and 300–458) comprising the linear neutralization sites of the G protein were spliced together in tandem by PCR-based site-directed mutagenesis and heterologously expressed in Escherichia coli (DE3). The recombinant protein (designated rRVg) was purified under denaturing conditions and solubilized by stepwise dialysis against an alkaline buffer (0.05M Na2CO3 pH 9.6). Western blot analysis of the antigenicity of rRVg showed that it was recognized by rabies-immune serum. Competitive neutralization assay revealed that rRVg significantly reduced the RV-neutralizing activity of the rabies-immune serum. These results show potential of rRVg as a diagnostic antigen for detecting RV-neutralizing antibodies in immunized hosts. [Copyright &y& Elsevier]

Abstract: Intelectin family, also called the X-lectin family, is a newly discovered gene family involved in development and innate immunity. However, no research was carried out for this gene family in the model organism zebrafish. Here we present the first characterization of seven zebrafish intelectins (zINTLs) and the first systematic comparative analysis of intelectins from various species in order to provide some clues to the function and evolution of this gene family. We examined the expression patterns of zINTLs in various development stages, normal adults, and Aeromonas salmonicida infected adults. Results showed that zINTL1–3 were highly expressed in one or several adult tissues. zINTL4–7, however, were expressed at quite low levels both in adults and various development stages, and some of them showed relaxation of functional constrains as revealed by K a/K s calculation. Of the seven zINTLs, zINTL3 was expressed predominantly in the liver and highly up-regulated upon infection, suggesting its important roles in immunity. Based on the characterization of zebrafish intelectins, we then conducted a systematic survey of intelectin members in various species and made comparative analyses. We found out that intelectin family may be a deuterostome specific gene family; and their expression patterns, quaternary structures and glycosylations vary considerably among various species, though their sequences are highly conserved. Moreover, these varied features have evolved multiple times independently in different species, resulting in species-specific protein structures and expression patterns. [Copyright &y& Elsevier]