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Serine hydroxymethyltransferase (SHMT) plays a vital role in one-carbon metabolic, photorespiration, and various stress responses. However, the genome-wide analysis has not been performed in wheat. In this study, a total of 12 TaSHMT genes were identified in wheat and classified into groups Ⅰa, Ⅰb, and Ⅱb. TaSHMT genes in each group shared similar conserved domain distributions. Chromosomal location, synteny, and cis-elements analysis of TaSHMTs were also analyzed. Real-time PCR results indicated that most TaSHMT genes were mainly expressed in leaves and stems during the wheat seedling stage. Most TaSHMT genes could respond to various abiotic stress. The growth of yeast cells expressing TaSHMT2.1 was inhibited under salt and dehydration stress. Moreover, the gene ontology (GO) annotation and protein interaction of TaSHMT genes were analyzed. These results increase our understanding of SHMT genes and provide robust candidate genes for further functional investigations aimed at crop improvement.

Rationale: Recurrent and metastatic cancers often undergo a period of dormancy, which is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the survival of quiescent cancer cells remain obscure. Methods: Dual genome-editing was carried out using a CRISPR/Cas9-based system to label endogenous p27 and Ki67 with the green and red fluorescent proteins EGFP and mCherry, respectively, in melanoma cells. Analysis of transcriptomes of isolated EGFP-p27highmCherry-Ki67low quiescent cells was conducted at bulk and single cell levels using RNA-sequencing. The extracellular acidification rate and oxygen consumption rate were measured to define metabolic phenotypes. SiRNA and inducible shRNA knockdown, chromatin immunoprecipitation and luciferase reporter assays were employed to elucidate mechanisms of the metabolic switch in quiescent cells. Results: Dual labelling of endogenous p27 and Ki67 with differentiable fluorescent probes allowed for visualization, isolation, and analysis of viable p27highKi67low quiescent cells. Paradoxically, the proto-oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27highKi67low quiescent cells and supported their survival through promoting mitochondrial oxidative phosphorylation (OXPHOS). In this context, c-Myc selectively transactivated genes encoding OXPHOS enzymes, including subunits of isocitric dehydrogenase 3 (IDH3), whereas its binding to cell cycle progression gene promoters was decreased in quiescent cells. Silencing of c-Myc or the catalytic subunit of IDH3, IDH3α, preferentially killed quiescent cells, recapitulating the effect of treatment with OXPHOS inhibitors. Conclusion: These results establish a rigorous experimental system for investigating cellular quiescence, uncover the high selectivity of c-Myc in activating OXPHOS genes in quiescent cells, and propose OXPHOS targeting as a potential therapeutic avenue to counter cancer cells in quiescence.

Objectives Adipocytes and adipocyte lipid metabolism are closely related with obesity and type 2 diabetes, but the molecular mechanism still needs further investigation. The aim of this study is to discover the adipocyte genes and pathways involved in obesity and type 2 diabetes using bioinformatics analysis. Methods The GSE27951 gene expression profile was obtained. Software and online tools (STRING, Cytoscape, BioGPS, CTD, and FunRich) were used to identify core genes.21 human subcutaneous adipose samples, with 10 from type 2 diabetic patients and 11 from normal controls, were included in these analyses. Results 184 differentially expressed genes (DEGs) including 42 up-regulated genes and 142 down-regulated genes were found to be enriched in metabolism, receptor activity, collagen type IV and glutamine biosynthesis I pathway by using the enrichment analysis. Seven hub genes were identified from the PPI network using various software (Cytoscape, STRING, BioGPS, and CTD). Four core genes (COL4A2, ACACB, GLUL, and CD36) were found to be highly expressed in subcutaneous adipose tissue of obese patients accompanying type 2 diabetes. Conclusion COL4A2, ACACB, GLUL and CD36 might be the core molecular biomarkers of obesity in patients with or without type 2 diabetes.

Acute influenza infection has been reported to be associated with neurological symptoms such as influenza-associated encephalopathy (IAE). Although the pathophysiology of this condition remain unclear, neuroinflammation and associated alterations in the central nervous system (CNS) are usually induced. Microglia (MGs), CNS-resident macrophages, are generally the first cells to be activated in response to brain infection or damage. We performed reverse transcriptase droplet digital PCR (RT-ddPCR) and luminex assays to investigate virus proliferation and immune reactions in BV2 MGs infected with influenza A(H1N1)pdm09 virus. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to investigate the dynamic change in the protein expression profile in BV2 MGs to gain insight into the CNS response to influenza A (H1N1) pdm09 infection. Our results showed that the influenza A(H1N1)pdm09 virus was replicative and productive in BV2 MG cells, which produced cytokines such as interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. The expression of osteopontin (OPN) in the influenza A (H1N1) pdm09-infected BV2 MGs was upregulated at 16 and 32 h post-infection (hpi) compared to that in the control group, resulting in aggravated brain damage and inflammation. Our study indicates that OPN signalling might provide new insights into the treatment of CNS injury and neurodegenerative diseases in IAE.

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) holds promising potential to generate functional cardiomyocytes for drug development and clinical applications, especially for direct in situ heart regeneration by delivery of reprogramming genes into adult cardiac fibroblasts in injured hearts. For a decade, many cocktails of transcription factors have been developed to generate iCMs from fibroblasts of different tissues in vitro and some were applied in vivo. Here, we aimed to develop genetic cocktails that induce cardiac reprogramming directly in cultured cardiac fibroblasts isolated from adult mice with myocardial infarction (MICFs), which could be more relevant to heart diseases. We found that the widely used genetic cocktail, Gata4, Mef2c, and Tbx5 (GMT) were inefficient in reprogramming cardiomyocytes from MICFs. In a whole well of a 12-well plate, less than 10 mCherry+ cells (Myh6-reporter transgenic MICFs. By screening 22 candidate transcription factors predicted through analyzing the gene regulatory network of cardiac development, we found that five factors, GMTMS (GMT plus Myocd and Sall4), induced more iCMs expressing the cardiac structural proteins cTnT and cTnI at a frequency of about 22.5 ± 2.7% of the transduced MICFs at day 21 post infection. What is more, GMTMS induced abundant beating cardiomyocytes at day 28 post infection. Specifically, Myocd contributed mainly to inducing the expression of cardiac proteins, while Sall4 accounted for the induction of functional properties, such as contractility. RNA-seq analysis of the iCMs at day 28 post infection revealed that they were reprogrammed to adopt a cardiomyocyte-like gene expression profile. Overall, we show here that Sall4 and Myocd play important roles in cardiac reprogramming from MICFs, providing a cocktail of genetic factors that have potential for further applications in in vivo cardiac reprogramming.

Accumulating evidence suggests that radiation treatment causes an adaptive response of lung adenocarcinoma (LUAD), which in turn attenuates the lethal effect of the irradiation. Previous microarray assays manifested the change of gene expression profile after irradiation. Bioinformatics analysis of the significantly changed genes revealed that VANGL1 may notably influence the effect of radiation on LUAD. To determine the role of VANGL1, this study knocked down or overexpressed VANGL1 in LUAD. M6A level of VANGL1 mRNA was determined by M6A-IP-qPCR assay. Irradiation caused the up-regulation of VANGL1 with the increase of VANGL1 m6A level. Depletion of m6A readers, IGF2BP2/3, undermined VANGL1 mRNA stability and expression upon irradiation. miR-29b-3p expression was decreased by irradiation, however VANGL1 is a target of miR-29b-3p which was identified by Luciferase report assay. The reduction of miR-29b-3p inhibited the degradation of VANGL1 mRNA. Knockdown of VANGL1 enhanced the detrimental effect of irradiation on LUAD, as indicated by more severe DNA damage and increased percentage of apoptotic cells. Immunocoprecipitation revealed the interaction between VANGL1 with BRAF. VANGL1 increased BRAF probably through suppressing the protein degradation, which led to the increase of BRAF downstream effectors, TP53BP1 and RAD51. These effectors are involved in DNA repair after the damage. In summary, irradiation caused the up-regulation of VANGL1, which, in turn, mitigated the detrimental effect of irradiation on LUAD by protecting DNA from damage probably through activating BRAF/TP53BP1/RAD51 cascades. Increased m6A level of VANGL1 and reduced miR-29b-3p took the responsibility of VANGL1 overexpression upon irradiation.

Long non-coding RNAs (lncRNAs) are closely associated with tumorigenesis of various malignancies, including glioma. However, the roles of most lncRNAs in glioma remain undiscovered. The present study for the first time explored the roles of NFIA-AS2 in glioma. Based on informatic analyses by online database, lncRNA NFIA-AS2 in glioma tissues was overexpressed and further confirmed in glioma tissues and cells by quantitative real-time PCR (qRT-PCR). High expression of NFIA-AS2 was closely correlated with poor prognosis and might be an independent prognostic factor for PFS and OS. Functionally, silenced NFIA-AS2 could remarkably hinder glioma cell proliferation, migration and invasion, and cause the apoptosis. Mechanistic investigation disclosed that NFIA-AS2 interacted with miR-655-3p and inversely connected with miR-655-3p in glioma. Additionally, miR-655-3p was proved to regulate the expression of ZFX. Final rescue assay demonstrated that ZFX overexpression or miR-655-3p downregulation could neutralize the suppressive effects of NFIA-AS2 knockdown on glioma progression. In conclusion, this study firstly reported that NFIA-AS2 could promote the progression of glioma by targeting the miR-665-3p/ZFX axis, which highlighted that NFIA-AS2 could be a novel biomarker and therapeutic target for glioma patients.

Programmed death 1 (PD-1) has been reported to be associated with aberrant regulation of T cells activation in aplastic anemia (AA). However, the connection between PD-1 expression status and AA needs to be further explored. The aim of this study is to investigate PD-1 expression status on T cells in AA patients and to explore the effect of PD-1 on apoptosis of T cells and BMHSCs. The concentration of platelet, lymphocyte and hemoglobin in peripheral blood of AA patients and healthy volunteers was detected by automatic blood-counter system. Mononuclear cells (MNCs) of peripheral blood and marrow were isolated from AA patients and healthy volunteers. PD-1 expression level on CD3+, CD4+, CD8+, CD4+CD25+FOXP3+ T cells and bone marrow hematopoietic stem cells (BMHSCs) and B7-H1 expression level on peripheral blood mononuclear cells (PBMCs) and BMHSCs were detected by flow cytometry (FCM). Cell apoptosis on T cells and BMHSCs was also detected by FCM. PD-1, B7-H1, Bax and Bcl-2 mRNA expression levels on T cells of peripheral blood were detected by real-time PCR. MNCs from healthy volunteers were treated with ammonium pyrrolidine dithiocarbamate (PDTC) to block the nuclear factor (NF)-кB pathway. Our results showed that in AA patients, T cells had high levels of PD-1 expression and apoptosis rate. In BMHSCs, apoptosis rate was also higher than healthy volunteers. The expression of PD-1 on T cells was correlated with lymphocyte count and hemoglobin level. PD-1 was highly expressed on CD4+CD25+FOXP3+ T cells of AA patients and PD-1 expression was negatively correlated with the percentage of CD4+CD25+FOXP3+ T cells in AA patients. B7-H1, the ligand of PD-1, was also highly expressed on T cells, B cells, PBMCs and BMHSCs. When the NF-κB pathway was blocked by PDTC, the apoptosis of T cells and BMHSCs was reduced in a dose-dependent manner and PD-1 expression was also decreased in a dose-dependent manner. In conclusion, PD-1 was up-regulated in T cells in AA patients compared with healthy volunteers. The NF-κB pathway participated in the process of apoptosis of CD4+CD25+FOXP3+ T cells and BMHSCs induced by PD-1 in AA patients.

Iodine antiseptics exhibit superior antimicrobial efficacy and do not cause acquired microbial resistance. However, they are underused in comparison with antibiotics in infection treatments, partly because of their adverse effects such as pain and allergy. The cause of these noxious effects is not fully understood, and no specific molecular targets or mechanisms have been discovered. In this study, we show that iodine antiseptics cause pain and promote allergic contact dermatitis in mouse models, and iodine stimulates a subset of sensory neurons that express TRPA1 and TRPV1 channels. In vivo pharmacological inhibition or genetic ablation of these channels indicates that TRPA1 plays a major role in iodine antiseptics‐induced pain and the adjuvant effect of iodine antiseptics on allergic contact dermatitis and that TRPV1 is also involved. We further demonstrate that iodine activates TRPA1 through a redox mechanism but has no direct effects on TRPV1. Our study improves the understanding of the adverse effects of iodine antiseptics and suggests a means to minimize their side effects through local inhibition of TRPA1 and TRPV1 channels.

Introduction: Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells (PCs) characterized by the clonal expansion and accumulation of monotypic PCs in the bone marrow (BM). Despite the remarkable improvements have been done in knowledge on the pathobiology of MM, therapy and medical care, MM still remains an incurable disease. Therefore elucidation of regulatory circuits altered such as long non-coding RNAs (lncRNAs) will allow us to obtain a better understanding into the biology and targeted therapy of MM. Overwhelming evidence suggests that lncRNAs have huge impact on adjusting gene expression through the processes of transcription and post-transcription regulation, genomic imprinting, and chromatin modification. In our previous study, we validated one lncRNA named ST3 beta-galactoside alpha-2,3-sialyltransferase 6 antisense RNA 1 (ST3GAL6-AS1) which was upregulated markedly in MM patients. Further research revealed that ST3GAL6-AS1 promote the adhesion, migration and invasion ability of MM cells. To determine the precise mechanism underlying ST3GAL6-AS1 biological function, we expound its interactive protein in this study. Methods: Comprehensive Identification of RNA binding Proteins by Mass Spectrometry (ChIRP-MS) was utilized to identify interactive protein of ST3GAL6-AS1. MM cell line MM.1S was reversibly crosslinked with UV/formaldehyde under native condition, with or without prior lysis, then hybridized with biotinylated DNA antisense and pulled down with streptavidin dynabeads. Peptides were identified by MS eventually. Raw MS files were processed with MaxQuant (Version 1.5.6.0). Both peptide and protein FDR should be less than 0.01. Gene Ontology (GO) analysis (http://david.abcc.ncifcrf.gov/summary.jsp) was used to analyze differentially expressed transcripts. Pathway analysis of differentially expressed transcripts was accomplished using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg). String online software (http://www.string-db.org/) was utilized to predict protein-protein interactions. Results: ChIRP-MS analysis identified a total of 599 proteins were interacted with ST3GAL6-AS1, among them, 177 proteins were identified to bind with ST3GAL6-AS1 specifically compared with control group (Figure 1A). GO and KEGG analysis were based on these 177 specific proteins. GO analysis showed that the top ten enriched biological process (BP) were mainly involved in mRNA catabolic process and splicing, while cellular component (CC) was cytosolic part and molecular function (MF) was structural constituent of ribosome (Figure 1B). KEGG pathway analysis of the top five enriched pathways included spliceosome and ribosome (Figure 1C). In the specific interaction proteins, we found multiple proteins belong to heterogeneous nuclear ribonucleoproteins (hnRNP) family, such as hnRNPA, hnRNPU. Further research showed that involved hnRNPs interacted with each other (Figure 1D) and mainly took important role in spliceosome by GO and KEGG analysis (FDR Conclusions: We have identified a group of proteins in MM cells which were specifically interacted with lncRNA ST3GAL6-AS1. These proteins mainly participate in mRNA catabolism and spliceosome, which might was the mechanism of ST3GAL6-AS1 to regulate its target gene in MM. Further investigation will focus on the exact action of ST3GAL6-AS1 through its interacting proteins. Figure 1 Disclosures No relevant conflicts of interest to declare.

The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversification occurred in the promoter regions and the exon-intron structures of the two Pks paralogs (Pks1 and Pks2). These two Pks genes have distinct expression patterns, with Pks1 highly expressed during conidiation and Pks2 highly expressed during infection. Different upstream signaling pathways were found to regulate the two Pks genes. Pks1 is positively regulated by Hog1-MAPK, Slt2-MAPK and Mr-OPY2, while Pks2 is positively regulated by Fus3-MAPK and negatively regulated by Mr-OPY2. Pks1 and Pks2 have been subjected to positive selection and synthesize different secondary metabolites. PKS1 is involved in synthesis of an anthraquinone derivative, and contributes to conidial pigmentation, which plays an important role in fungal tolerance to UV radiation and extreme temperatures. Disruption of the Pks2 gene delayed formation of infectious structures and increased the time taken to kill insects, indicating that Pks2 contributes to pathogenesis. Thus, the duplication of a Pks gene cluster and its subsequent functional diversification has increased the adaptive flexibility of Metarhizium species., Author summary In fungi, gene clusters that synthesize secondary metabolites are hotspots for the generation of fungal metabolic diversity through gene duplication, but their ecological importance remains poorly understood. Metarhizium species are adapted to life as insect pathogens, plant symbionts and saprophytes, enabling the function of individual genes to be studied in diverse fungal lifestyles. We discovered that a duplication of a Pks (polyketide synthase) gene cluster in Metarhizium species has facilitated its ecological opportunism. Sequence diversifications occurred in the promoter regions, the intro-exon structures, and the coding sequences of the two Pks paralogs, and they synthesize different secondary metabolites, have different expression patterns, and are regulated by different signaling pathways. PKS1s involved in synthesis of conidial pigments and tolerance to several abiotic stresses. The Pks2 gene is involved in formation of infectious structures (appressoria), enabling these fungi to kill insects faster. This Pks gene cluster duplication event may have been important for the adaptation of Metarhizium species to diverse environments.

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.

Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070bp, containing an open reading frame of 555bp. The deduced amino acid sequence shared high similarity (45.1-67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in immune system. In the present study, the IL-10 gene of African clawed frog (Xenopus tropicalis) was first cloned, and its expression pattern and 3D structure were also analyzed. The frog IL-10 mRNA encoded 172 amino acids which possessed several conserved features found in IL-10s from other species, including five-exon/four-intron genomic structure, conserved four cysteine residues, IL-10 family motif, and sixα-helices. Real-time PCR showed that frog IL-10 mRNA was ubiquitous expressed in all examined tissues, highly in some immune related tissues including kidney, spleen, and intestine and lowly in heart, stomach, and liver. The frog IL-10 mRNA was upregulated at 24 h after LPS stimulation, indicating that it plays a part in the host immune response to bacterial infection. Another IL, termed as IL-20, was identified from the frog IL-10 locus, which might be the homologue of mammalian IL-19/20 according to the analysis results of the phylogenetic tree and the sequence identities.

Plant 14-3-3 proteins belong to a large family of proteins involved in numerous physiological processes, and function by binding to phosphorylated client proteins to regulate their function. However, little is known about their regulatory mechanisms in rice root growth. In this study, four 14-3-3 isoforms (GF14b, GF14c, GF14e, GF14f) exhibiting prominent expression profiles in rice roots, were selected for further investigation. Through a pull-down assay using four 14-3-3 isoforms in rice roots, 87 client proteins were identified that are involved in metabolism, protein synthesis and trafficking, energy metabolism, cell structure and growth, and other cellular processes. Importantly, we found that 14-3-3 proteins may play an important role in the root stress response through interactions with proteins functioning in oxidative stress, pathogenesis and secondary metabolism. By using real-time RT–PCR, it was found that 14-3-3 proteins exhibited diverse patterns of gene expression in response to salinity and drought stresses in rice root. The results revealed the isoform-specific functions of root 14-3-3 proteins. Our current study provides insight into understanding the functional roles of 14-3-3 proteins during rice root growth. Significance Rice ( Oryza sativa L.) is one of the most important food crops in the world, as it is consumed by more than 3 billion people. Regulation of root growth plays an important role in rice adaption to biotic and abiotic, stress such as rhizosphere microbes and nutrient stress, and this process is directly related to the final yield. 14-3-3 proteins form a multi-gene family regulating developmental processes in plants. However, the correlation between the 14-3-3 protein family and its role in rice root growth has very little study. We applied an affinity chromatographic approach, in combination with LC-MS/MS, to explore the client proteins of four 14-3-3 isoforms that exhibit much more prominent gene expression than other members of the 14-3-3 family in rice roots. Assessments of the identified client proteins are able to obtain novel information toward understanding the functional mechanism of 14-3-3 proteins in rice root growth as expected.

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose-derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co-cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co-cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co-culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up-regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP-1, TGF-β1, IGF-1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up-regulated. Collectively, these results suggest that direct co-cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD.

Purpose: The aim of this study was to investigate the relationship between the expressions of microRNA-9 (miR-9) and microRNA-200c (miR-200c) in human breast cancers and clinicopathological features. Methods: We investigated the expressions of miR-9 and miR-200c in 68 patients with breast cancers using the quantitative reverse transcription–polymerase chain reaction method, and assessed the E-cadherin status using the immunohistochemistry method. Results: The relative expression levels of miR-9 and miR-200c in breast cancer patients with lymph node metastasis were higher than that of patients without lymph node metastasis. The expression of miR-9 correlated inversely with E-cadherin expression. Conclusions: The results showed that higher expressions of miR-9 and miR-200c in human breast cancers were associated with lymph node metastasis. This study indicated that the elevation of miR-9 and miR-200c in human breast cancers can induce an invasive phenotype and may serve as a molecular diagnostic ma...

Corticotropin releasing factor (CRF), a peptide hormone involved in the stress response, holds a key position in cardiovascular regulation. Here, we report that the central effect of CRF on cardiovascular activities is mediated by the posterior hypothalamic nucleus (PH), an important structure responsible for stress-induced cardiovascular changes. Our present results demonstrate that CRF directly excites PH neurons via two CRF receptors, CRFR1 and CRFR2 and consequently increases heart rate (HR) rather than the mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). Bilateral vagotomy does not influence the tachycardia response to microinjection of CRF into the PH, while β adrenergic receptor antagonist propranolol almost totally abolishes the tachycardia. Furthermore, microinjecting CRF into the PH primarily increases neuronal activity of the rostral ventrolateral medulla (RVLM) and rostral ventromedial medulla (RVMM), but does not influence that of the dorsal motor nucleus of the vagus nerve (DMNV). These findings suggest that the PH is a critical target for central CRF system in regulation of cardiac activity and the PH-RVLM/RVMM-cardiac sympathetic nerve pathways, rather than PH-DMNV-vagus pathway, may contribute to the CRF-induced tachycardia.

Ubiquitin-conjugating enzyme E2D3 (UBE2D3), a key component in ubiquitin (Ub) proteasome system, plays a crucial role in tumorigenesis. We previously found that it is bound to hTERT, and UBE2D3 could attenuate radiosensitivity of human breast cancer cells. Here we investigated a contributing role of UBE2D3 in radiosensitivity of esophageal squamous carcinoma. We demonstrated that the overexpression of UBE2D3 in esophageal squamous carcinoma cells (EC109) resulted in prolonged G1 phase and shortened G2/M phase after irradiation. UBE2D3 overexpression also decreased length of telomere and activity of telomerase. In addition, the overexpression of UBE2D3 increased mRNA expression but decreased protein levels of hTERT in both vitro and vivo systems. Compared with untreated cells, the treatment of UBE2D3 overexpressing cells with the specific proteasome inhibitor (MG132) could up-regulate hTERT. MG132 treatment of UBE2D3 overexpressed cells caused a clear and dramatic increase in the amount of ubiquitinated hTERT species. These findings indicate that UBE2D3 enhances radiosensitivity of EC109 cells by degradating hTERT through the ubiquitin proteolysis pathway.

MicroRNAs (miRNAs) play an important role in the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms including nervous system. However, the role of miRNAs in primary neurons of dorsal root ganglion (DRG) after injury was not clear. In this study, a miRNA microarray analysis was performed, and a total of 21 miRNAs were found to be down-regulated following unilateral sciatic nerve transection. The miR-144, miR-145, and miR-214 were further validated using quantitative reverse transcriptase PCR (qRT-PCR). Moreover, in situ hybridization (ISH) experiments using locked nucleic acid (LNA)-modified DNA oligonucleotide probes verified that miR-144, miR-145, and miR-214 were expressed in primary neurons of DRG and down-regulated following sciatic nerve transection. Predictions of potential miRNA targets involved were identified by performing a bioinformatics analysis. These predictions were tested using miRNA luciferase reporter vectors, with Robo2 and srGAP2 evaluated as the potential targets of miR-145 and miR-214, respectively. The role of miR-145 in cultured primary neurons was also investigated, and the result found that miR-145 miR-145 inhibited neurite growth and down-regulated Robo2 expression. Finding from this study suggested that miRNAs of DRG can mediated the course of regeneration including through Slit-Robo-srGAP signaling pathway after injury.

Suaeda salsa calluses cultured in darkness for 28 d were transferred to Murashige and Skoog (MS) media containing various growth regulators under white light conditions for 10 d to investigate cell growth, betacyanin accumulation, and expression of dopa-4,5-dioxygenase (DODA). Callus growth was markedly promoted when 0.2 mg·L−1 2,4-D and 0.5 mg·L−1 6-BA were added to the MS medium. Surprisingly, of the auxins tested, IAA had no effect on betacyanin content, but 2,4-D strongly decreased betacyanin content. Betacyanin content was positively correlated with 6-BA concentrations in the range of 0.1–2.0 mg·L−1. DODA mRNA levels were consistent with the response of betacyanin content to exogenous growth regulators. These results suggest that betacyanin metabolism in S. salsa calluses is regulated under white light conditions by growth regulators through the regulation of genes such as DODA that are involved in betacyanin synthesis.

Context: Progesterone and its receptor (PR) play key roles in uterine leiomyoma growth. Previously, using chromatin immunoprecipitation-based cloning, we uncovered L-type amino acid transporter 2 (LAT2) as a novel PR target gene. LAT2 forms heterodimeric complexes with 4F2 heavy chain (4F2hc), a single transmembrane domain protein essential for LAT2 to exert its function in the plasma membrane. Until now, little is known about the roles of LAT2/4F2hc in the regulation of the growth of human uterine leiomyoma. Objective: The aim of the study is to investigate the regulation of LAT2 and 4F2hc by progesterone and the antiprogestin mifepristone and their functions in primary human uterine leiomyoma smooth muscle (LSM) cells and tissues from 39 premenopausal women. Results: In primary LSM cells, progesterone significantly induced LAT2 mRNA levels, and this was blocked by cotreatment with mifepristone. Progesterone did not alter 4F2hc mRNA levels, whereas mifepristone significantly induced 4F2hc mRNA expression. Small interfering RNA knockdown of LAT2 or 4F2hc markedly increased LSM cell proliferation. LAT2, PR-B, and PR-A levels were significantly higher in freshly isolated LSM cells vs. adjacent myometrial cells. In vivo, mRNA levels of LAT2 and PR but not 4F2hc were significantly higher in leiomyoma tissues compared with matched myometrial tissues. Conclusion: We present evidence that progesterone and its antagonist mifepristone regulate the amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells. Our findings suggest that products of the LAT2/4F2hc genes may play important roles in leiomyoma cell proliferation. We speculate that critical ratios of LAT2 to 4F2hc regulate leiomyoma growth.

Thyroid hormone responsive spot 14 (THRSP), expressed in lipogenic tissues, is suggested as a transcription factor to regulate gene expression of rate-limiting enzymes in lipogenesis. Two THRSP isoforms, THRSPa and THRSPb, were detected at cDNA levels in chickens and ducks. Chicken THRSPa was speculated to be associated with growth development and lipid metabolism because of significant correlation between the indels in the coding region of THRSPalpha and growth, as well as abdominal fat traits of chickens. The differences of THRSP structure and expression between chickens and ducks were reviewed. Furthermore, polymorphism and genetic effects of THRSP gene in chickens and ducks were analyzed.

The adenylyl cyclase-associated protein (CAP) was first identified in the budding yeast as a protein that functions in cytoskeletal organization and signal transduction. We isolated two CAP homologs from wild-type cotton Gossypium arboreum L. (DPL971) and its natural fuzzless mutant (DPL972). Alignments of 3998-bp genomic and 1425-bp cDNA sequences revealed that the cap genes contain nine introns and an open reading frame (ORF) encoding a 471 amino acid protein. The predicted amino acid sequence shares high identity (∼70%) with Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Expression profiles of the gene were studied from ovule, leaf, hypocotyl, and root tissues, through reverse transcriptase polymerase chain reaction and Northern blotting analysis. The results revealed that the gene was expressed in all tissues tested in wild-type and fuzzless plants. The expression of GaCAP reached the highest level in both wild-type and mutant fibers during 1 to 4 d postanthesis (DPA), then gradually declined in the mutant after 4 DPA. By comparing the predicted amino acid sequences, a polar substitution was observed at a conserved position between wild-type and mutant. At this position, the conserved uncharged unpolar alanine (amino acid 44) of GaCAP was replaced by a polar threonine in GaCAPm. Therefore, our results suggest that GaCAP may play a functional role during early stages of cotton fiber development.

Objective The aim of this study was to examine the changes of ATF4 expression in cultured human periodontal ligament fibroblasts (hPDLF) after mechanical stimuli, and to investigate whether ATF4 is essential for the mechanical stress-induced hPDLF differentiation. Methods Reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting were used to examine mRNA and protein levels of ATF4 expression in hPDLFs after application of centrifugal force. pMyc-ATF4 transfected cells were subjected to centrifugal force for 30 min, and the changes of alkaline phosphatase (ALP) activity and osteocalcin (OCN), osteopontin (OPN), collagen I (COLI), bone sialoprotein (BSP) genes were measured to assess the differentiation of hPDLFs. Results The mRNA and protein levels of ATF4 increased shortly and then decreased rapidly towards its pre-pressure levels. Overexpression of pMyc-ATF4 exhibited a greater increase in ALP activity and all four osteogenic genes compared to the untransfected cells in response to the centrifugal force. Conclusion Our results indicate that ATF4 is essential in response of hPDLFs to mechanical stress, resulting in increased differentiation of hPDLFs to osteoblast-like cells.

The study was aimed to explore the underlying mechanisms and identify the potential target genes and key miRNAs for acute myeloid leukemia (AML) treatment by bioinformatics analysis. The microarray data of GSE9476 were downloaded from Gene Expression Omnibus database. A total of 64 samples, including 26 AML and 38 normal samples, were used to identify differentially expressed genes (DEGs) between AML and normal samples. The functional enrichment analysis was performed, and protein-protein interaction (PPI) network of the DEGs was constructed by Cytoscape software. Besides, the target miRNAs for DEGs were identified. Totally, 323 DEGs were identified, including 87 up-regulated and 236 down-regulated genes. Not only up-regulated genes but also down-regulated genes were related to hematopoietic-related functions. Besides, down-regulated genes were also enriched in primary immunodeficiency pathway. Tumor necrosis factor (TNF), interleukin 7 receptor (IL7R), lymphocyte-specific protein tyrosine kinase (LCK), CD79a molecule and immunoglobulin-associated alpha (CD79A) were identified in these functions. TNF and LCK were hub nodes in PPI networks. miR-124 and miR-181 were important miRNAs in this study. The hematopoietic-related functions and primary immunodeficiency pathway may be associated with AML development. Genes, such as TNF, IL7R, LCK and CD79A, may be potential therapeutic target genes for AML, and miR-124 and miR-181 may be key miRNAs in AML development.

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21 WAF1/CIP1 (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5′ end of the antisense strand, or “seed” sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.

Repeated electroacupuncture (EA) stimulation is known to enhance the circulating level of estrogen in the ovariectomized rats by stimulating the activity of the hypothalamus-pituitary-adrenal axis. To explore the mechanism of EA in promoting the homeostasis in ovariectomized rats, the hypothalamic CRH content and its mRNA expression as well as the circulating estradiol in the rats along with time after ovariectomy were detected. Female rats were divided into fourteen groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M; intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M; OVX1M with EA (OVX1M+EA) and intact 1 month with EA (INT1M+EA). The blood concentrations of estradiol (E2) and total contents of hypothalamic CRH were measured by radioimmunoassay. The expressions of CRHmRNA were semiquantified by RT-PCR. The results showed that the blood E(2) concentrations increased gradually along with time after ovariectomy in the rats. The level was higher in OVX1M+EA than those in OVX1M, 2M, 3M (p < 0.05), which was near to the level in OVX6M. The total content (ng/g tissue) of hypothalamic CRH and the CRHmRNA expressions in the OVX6M rats were higher than other groups (p < 0.05). The CRH content or mRNA expression in the OVX1M+EA increased significantly compared with that in OVX1M (p < 0.05), and it was close to the level in the OVX4M and OVX5M. The results suggested that the circulating E2 and hypothalamic CRH enhances along with time after ovariectomy in rats, and the EA stimulation might speedup the process.

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation. Mol. Reprod. Dev. 71: 129–139, 2005. © 2005 Wiley-Liss, Inc.

STAT3 regulates many target genes in response to cytokines and growth factors. To study the mechanisms of STAT3-dependent transcription, we established several cell lines in which HepG2-STAT3-knockdown cells were reconstituted with a variety of STAT3 mutants. Using these cell lines, we found that truncated STAT3(1–750), but not STAT3(1–761), could not recruit SRC-1/NcoA-1 and was not phosphorylated on Ser727. Furthermore, mutation of STAT3 L755 and F757 to alanines caused the loss of STAT3-dependent SRC-1 recruitment, leaving Ser727 phosphorylation intact. Consistent with this, the STAT3-L755A/F757A mutant showed no increase in acetylated histone H3 at Lys14 and a decreased level of RNA polymerase II recruited to the target gene promoter, although p300 recruitment and histone H4 acetylation were intact. This mutant also lost responsiveness to co-expressed SRC-1. Thus, the conserved STAT3 region from 752 to 761, called STAT3 CR2, plays critical roles in STAT3-dependent transcription by recruiting SRC-1 and allowing Ser727 phosphorylation.

We investigated the role of 14-3-3sigma protein in insulin-like growth factor-I (IGF-I) receptor signaling. It has been previously shown that 14-3-3sigma negatively regulates cell cycle especially in response to p53-sensitive DNA damage. In this study we demonstrated that 14-3-3sigma is a positive mediator of IGF-I receptor-induced cell proliferation. Treatment with IGF-I increased 14-3-3sigma mRNA and protein levels about 4-fold, in a time-dependent manner in MCF-7 breast cancer cells. Preincubation with the phosphoinositide 3'-kinase inhibitor LY294002 significantly reduced the effects of IGF-I on 14-3-3sigma gene expression in these cells, suggesting that this effect of IGF-I occurs via the phosphoinositide 3'-kinase pathway. 14-3-3sigma is induced by IGF-I in MCF-7 cells, which express wild-type p53, as well as in MCF-7 cells transfected with a small interference RNA targeting duplex that reduced p53 expression levels. These results suggest that IGF-I induces 14-3-3sigma expression in a manner that is independent of p53. Using the small interference RNA strategy, we demonstrated that a 70-75% reduction of 14-3-3sigma mRNA levels resulted in a similar decrease in the effects of IGF-I on cell cycle progression and proliferation in MCF-7 cells. This effect was also associated with a reduction in IGF-I-induced cyclin D1 expression. Taken together, these results suggest that 14-3-3sigma positively mediates IGF-I-induced cell cycle progression.

Leukocytes play an important role in the progression of disease and leukocyte-derived proteins are associated with the pathogenesis of the disease. Leukocyte activation causes production of inflammatory mediators, over-expression of cell surface adhesion molecules, and an increase in migration and infiltration, phagocytosis, and degranulation, as well as receptor phosphorylation and signal transduction. An increasing number of studies on the application of leukocyte proteomics have appeared in mapping protein profiles of inflammatory cells, contributing to the understanding of potential mechanisms involved in leukocyte function. Together with improvements in proteomic technology in leukocyte research, leukocyte proteomic analysis becomes more simple, rapid, flexible, sensitive, and specific. This enables proteomic investigation of activated or non-activated leukocytes to be highly focused on defined suborgans or specific signaling pathways. Research in leukocyte proteomics is progressing from fingerprinting to functioning, human cell lines to primary leukocytes, non-activated cells to inflammatory mediator-stimulated cells, in vitro culture to in vivo challenge, and animal models to human disease. A number of newly identified proteins from leukocyte proteomics may offer new mechanism-orientated targets for drug discovery and development.

To study differential gene expression in porcine skeletal muscle, a porcine complementary DNA (cDNA) macroarray was produced that contained 327 expressed sequence tags (EST) derived from whole embryo and adult skeletal muscle, and differential display PCR products from fetal and postnatal muscle. Total RNA from four muscle samples, 75- and 105-d fetal hind limb muscles, and 1- and 7-wk postnatal semitendinosus muscle was used to make radiolabeled targets for duplicate hybridization to the macroarray membranes in an initial screen for expression. All EST that gave clear signals (n = 238) were then re-arrayed, and hybridization was conducted with additional biological replication of samples in the 75-d and 1-wk ages. Signal intensity for each gene was normalized to signal intensity measured at control spots on each membrane, which consisted of total cDNA from liver, lung, spleen, and skeletal muscle. Both normalized ratio levels and a mixed linear model analyses were used to identify genes differentially expressed among the muscle samples. Results showed 28 genes had differences in expression level greater than twofold between the 75-d fetal and 1-wk muscle RNA samples. All 28 genes were also identified as genes with significantly different (P 100; P < 0.01) to known genes, two matched genes encoding human hypothetical proteins, and seven had no significant matches to Genbank nonredundant and dbEST (database of expressed sequence tags) entries. These results were confirmed for representative genes with RNA blot analysis of seven developmental time points, including RNA from the same muscle samples tested previously in the macroarray. The RNA blot results confirmed the macroarray results for all selected genes, demonstrating that the macroarray technique used in this study is accurate and reproducible. An unknown muscle clone (M218) with a slightly less than twofold increase in expression from the 75-d to the 1-wk age (1 wk/75 d = 1.94; P = 0.0114) was also shown to differ between these two ages using RNA blot analysis, demonstrating the methods used to identify differentially expressed genes may be conservative. The association between expression patterns of vimentin and desmin was also investigated. Results indicate the switch in intermediate filament protein from vimentin to desmin occurs primarily at the level of transcription and/or RNA processing.

The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54fold more highly than Ig-mutated CLL (P < 10-21). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Igunmutated CLLs had low ZAP-70 expression. Among these ZAP-70 “outliers,” those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase‐polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL. (Blood. 2003;101:4944-4951)

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

Early detection of cysteine rich protein 61 (CYR61, CCN1) in urine following renal ischemic reperfusion injury. Background Acute renal failure (ARF) has a high morbidity and mortality. Many therapies have worked in animals but were unsuccessful in clinical trials. The inability to diagnose ARF early may have impaired the success of these trials. Method We screened a subtraction library to search for potential disease markers that would be induced rapidly after renal injury. Mice and rats were subjected to 30 to 40 minutes of bilateral ischemia. Results mRNA for Cyr61 , a secreted growth factor-inducible immediate early gene, was markedly up-regulated at two hours in the kidney but not other organs following renal ischemia. In situ hybridization studies suggested Cyr61 was synthesized in the proximal straight tubule. Cyr61 protein was analyzed by capture with heparin beads followed by Western blotting. Induction of Cyr61 protein could be detected in the kidney within one hour, peaked at four to eight hours, and remained elevated for at least 24 hours following ischemia. Cyr61 protein was detected in urine at three to six hours and peaked at six to nine hours after renal injury. Cyr61 was not detected after volume depletion, which is often difficult to differentiate from ARF. Conclusions The secreted, cysteine-rich, heparin binding protein Cyr61 is rapidly induced in proximal straight tubules following renal ischemia, and excreted in the urine where it might serve as an early biomarker of renal injury.

To elucidate the physiological roles of endothelin-1 (ET-1) and endothelin-2 (ET-2)/vasoactive intestinal contractor (VIC) in gastric injury of mice, we measured the gene expression rates of ET-1 and ET-2/VIC in gastric injury induced by ethanol in young (8 weeks) and old (>33 weeks) mice. Mice that were fasted for 24h were injected with absolute ethanol intragastrically and killed after 1 or 4h of ethanol exposure. The size of the gastric lesions increased gradually after ethanol exposure and was at its greatest after 4 h, in both young and old mice. The gene expression of ET-1 tended to increase after 1h and to decrease by 4h of ethanol exposure in both young and old mice. However, the gene expression of ET-2/VIC in young mice increased significantly after 1 and 4h of ethanol exposure, whereas the gene expression of ET-2/VIC in the old mice did not change after ethanol exposure. Based on these results, we conclude that aging influences the gene expression of ET-2/VIC but not lesion size or gene expression of ET-1 in ethanol-induced gastric injury in the mouse. We therefore suggest that regulation of gene expression of these two genes differs during the course of aging.

Introduction Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. Methods iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. Results High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. Conclusions Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0527-x) contains supplementary material, which is available to authorized users.

Objective Beclin-1 has recently been observed as an essential marker of autophagy in several cancers. However, the prognostic role of Beclin-1 in colorectal neoplasia remains controversial. Our study aimed to evaluate the potential association between Beclin-1 expression and the outcome of colorectal cancer patients. Materials and methods All related studies were systematically searched in Pubmed, Embase, Springer and Chinese National Knowledge Infrastructure databases (CNKI), and then a meta-analysis was performed to determine the association of Beclin-1 expression with clinical outcomes. Finally, a total of 6 articles were included in our analysis. Results Our data showed that high Beclin-1 expression in patients with CRC was associated with poor prognosis in terms of tumor distant metastasis (OR=2.090, 95%CI=1.061-4.119, p=0.033) and overall survival (RR=1.422, 95%CI=1.032-1.959, p=0.031). However, we did not found any correlation between Beclin-1 over-expression and tumor differentiation (OR=1.711, 95%CI=0.920-3.183, p=0.090). In addition, there was no evidence of publication bias as suggested by Egger's tests for tumor distant metastasis (p=1.000), differentiation (p=1.000) and OS (p=0.308). Conclusions Our present meta-analysis indicated that elevated Beclin-1 expression iss associated with tumor metastasis and a poor prognosis in patients with CRC. Beclin-1 might serve as an efficient prognostic indicator in CRC, and could be a new molecular target in CRC therapy.

HER2 receptor dimerization is a critical step in the HER2 activation process. Here, we demonstrated that heat shock protein gp96 on cell membrane interacts with HER2, facilitates HER2 dimerization and promotes cell proliferation. Cell membrane gp96 levels were observed to correlate with HER2 phosphorylation in primary breast tumors. Finally, we provide evidence that targeting gp96 with a specific monoclonal antibody led to decreased cell growth and increased apoptosis in vitro, and suppression of tumor growth in vivo. Our work represents a new therapeutic strategy for inhibiting HER2 signaling in cancer.

This study investigated the mechanisms by which licochalcone B (LCB) inhibits the adhesion,invasion and metastasis of human malignant bladder cancer T24 cells. Cell viability was evaluated using a sulforhodamine B (SRB) assay. Cell migration and invasion ability were conducted using wound-healing assay and matrigel transwell invasion assay. The activities of matrix metalloproteinases (MMP)-2 and MMP-9 were measured by gelatin zymography protease assays. The expression in protein level of NF-κBP65 and AP-1 was determined using the ELISA method; the protein levels of MMP-9, NF-κBP65, IκBα and P-IκBα were detected by Western blot. The expression in mRNA level of MMP-9 was assessed using quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR. The results indicated that LCB attenuated T24 cell migration, adhesion and invasion in a concentration-dependent manner. LCB treatment down-regulated the mRNA expression, protein expression and activity of MMP-9 but had no effect on MMP-2. In addition, LCB treatment decreased the protein level of NF-кBP65 and nuclear translocation of NF-кB. These findings suggested that LCB attenuated migration of bladder cancer T24 cells and adhesion and invasion accompanied with down-regulated protein expression of MMP-9 and the nuclear translocation of NF-кB. Our results provide support that LCB may be a potent adjuvant therapeutic agent in the prevention and therapy of bladder cancer.

Background Litter size in pigs is a major factor affecting the profitability in the pig industry. The peri-implantation window in pigs is characterized by the coordinated interactions between the maternal uterine endometrium and the rapidly elongating conceptuses and represents a period of time during which a large percentage of the developing conceptuses are lost. However, the gene expression and regulatory networks in the endometrium contributing to the establishment of the maternal: placental interface remain poorly understood. Results We characterized the endometrial gene expression profile during the peri-implantation stage of development by comparing two breeds that demonstrate very different reproductive efficiencies. We employed the porcine Affymetrix GeneChip® to assay the transcriptomic profiles of genes expressed in the uterine endometrium obtained from Meishan and Yorkshire gilts (n = 4 for each breed) on day 12 of gestation (M12 and Y12, respectively). Total of 17,076 probesets were identified as "present" in at least two arrays. A mixed model-based statistical analysis predicted a total of 2,656 (q

Purpose: We studied the methylation status of E-cadherin gene promoter in prostate cancer and its relationship with E-cadherin inactivation in prostate cancer.Materials and Methods: Seven human prostate cell lines and 35 microdissected prostate cancer specimens were analyzed for E-cadherin promoter methylation using the bisulfite genome sequencing technique. E-cadherin messenger (m)RNA expression and protein expression were also studied in prostate cell lines by reverse transcriptase-polymerase chain reaction and in prostate cancer specimens by immunostaining, respectively.Results: The overall methylation of E-cadherin promoter was evident in 14 of 20 grades III to V (70%) and in 5 of 15 grades I to II (33%) prostate cancer samples. It correlated with absent or reduced E-cadherin immunostaining. Methylation in low grade tumors was present mainly in the exon region, whereas in high grade tumors methylation was also present in the promoter region. Methylation was noted in 2 of 6 prostate cancer cell lines (...

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose-derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co-cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co-cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co-culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up-regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP-1, TGF-β1, IGF-1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up-regulated. Collectively, these results suggest that direct co-cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD.

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.