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Shell formation is a dynamic process involving organic matrix secretion and calcification. In this study, we characterized shell morphogenesis during larval development in Crassostrea gigas. Using scanning electron microscopy (SEM) and fluorescence staining, we demonstrated that shell field, the first morphologically distinguishable shell-forming tissue, became visible soon after enlargement of the blastopore at the anterior end of the trochophore. Shell organic matrix namely protein polysaccharides and calcified structure appeared as a slit at the dorsal side of the embryo. The early shell field began to extend along the dorsal side of the trochophore larvae, and became a saddle shaped shell field that gave rise to the prodissoconch I embryonic shell in the early D-shaped larvae. Subsequently, prodissoconch II shell was formed in the late D-shaped larvae with a characteristic appearance of growth lines. To identify gene expression markers for studying shell formation, we isolated three potential larval shell formation genes CgPOU2F1 , CgSox5 , and CgPax6 and analyzed their expression during shell morphogenesis. The three potential shell formation genes possessed a similar pattern of expression. Their expression was detected in the shell gland and shell field regions in early D-shaped larvae, hereafter, their expression was detected at the larval mantle edge in the calcified shell stages. Together, these studies provide knowledge of shell morphogenesis in pacific oyster and molecular markers for studying the molecular regulation of biomineralization and shell formation. [Display omitted] • Molecular characteristics and gene expressions of POU2F1 , Sox5 , and Pax6 were studied in Crassostrea gigas. • CgPOU2F1 , CgSox5 , and CgPax6 were involved in larval shell formation. • Calcofluor white and calcein might be an ideal marker to visualize and trace the larval shell formation. [ABSTRACT FROM AUTHOR]

Background: Shell color polymorphisms of Mollusca have contributed to development of evolutionary biology and population genetics, while the genetic bases and molecular mechanisms underlying shell pigmentation are poorly understood. The Pacific oyster (Crassostrea gigas) is one of the most important farmed oysters worldwide. Through successive family selection, four shell color variants (white, golden, black and partially pigmented) of C. gigas have been developed. To elucidate the genetic mechanisms of shell coloration in C. gigas and facilitate the selection of elite oyster lines with desired coloration patterns, differentially expressed genes (DEGs) were identified among the four shell color variants by RNA-seq. Results: Digital gene expression generated over fifteen million reads per sample, producing expression data for 28,027 genes. A total number of 2,645 DEGs were identified from pair-wise comparisons, of which 432, 91, 43 and 39 genes specially were up-regulated in white, black, golden and partially pigmented shell of C. gigas, respectively. Three genes of Abca1, Abca3 and Abcb1 which belong to the ATP-binding cassette (ABC) transporters super-families were significantly associated with white shell formation. A tyrosinase transcript (CGI_10008737) represented consistent up-regulated pattern with golden coloration. We proposed that white shell variant of C. gigas could employ “endocytosis” to down-regulate notch level and to prevent shell pigmentation. Conclusion: This study discovered some potential shell coloration genes and related molecular mechanisms by the RNA-seq, which would provide foundational information to further study on shell coloration and assist in selective breeding in C. gigas. [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic β-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. This study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. A mouse-derived pancreatic islet β-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2−/−) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. Network pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro , FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2−/− mice. This study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics. [Display omitted] • Formononetin ameliorated alloxan-induced T1DM in wild-type C57BL/6J mice. • Formononetin could not ameliorated alloxan-induced T1DM in Nrf2−/− C57BL/6J mice. • Formononetin protected pancreatic islet β-cells from alloxan-induced cell injury. • Formononetin could bind to Keap1 to activate the Keap1/Nrf2 signaling pathway. [ABSTRACT FROM AUTHOR]

Chronic periodontitis is characterized by perturbation of the epithelial attachment to the tooth with subsequent migration of the lining epithelium and formation of a cleft or pocket. This non-keratinized lining epithelium provides initial responses to bacterial products by signalling through receptors of innate immunity to activate inflammasome pathways. These comprise an intracellular network of regulatory and effector molecules leading to synthesis and activation of pro-inflammatory cytokines. Conversely, CD24 is characteristically strongly expressed by the pocket epithelium and is reported to function as an important negative regulator for danger signals, protecting tissues from excessive leukocyte activity. The objective of the study was to determine the impact of ligation of CD24 on expression of inflammasome components. An epithelial mimic of pocket epithelium was used to evaluate activation of the NLRP3 inflammasome pathway. Surprisingly, antibody ligation of CD24 enhanced expression of NLRP3 together with co-activators ASC and caspase-1 resulting in burst release of activated interleukin (IL)-18. Potent product inhibition was detected with IL-18 suppressing expression of NLRP3, ASC, and caspase-1. Scant distribution of these products within pocket epithelium compared with healthy gingival attachment provided indication of potential cycling of NLRP3 inflammasome expression. As subjects with mild chronic periodontitis have increased titres of serum antibodies auto-reactive with CD24 compared with those of subjects with severe periodontitis, a molecular mechanism for regulated expression of the NLRP3 inflammasome mediated by c-Src kinase activity, is proposed. This pathway could be regionally disrupted by products of pathogenic bacteria with profound downregulation in the dysbiosis associated with severe disease. [ABSTRACT FROM AUTHOR]

• Carotenoid metabolism of Crassostrea gigas was studies for the first time with a beta-carotene feeding method. • The function of two apolipoprotein genes in carotenoid storage was validated by RNAi in the oyster. • A negative feedback regulation mechanism was proposed to regulate carotenoid metabolism in the oyster. Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster. [ABSTRACT FROM AUTHOR]

One of the most important physiological process in the life cycle of most bivalves is the sexual reproduction required for species propagation. Understanding the molecular regulatory mechanisms of gender determination and gonad development have potential application in bivalve aquaculture, but there is still limited knowledge about this issue. The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide, as well as a representative specie to study the sexual development in bivalves for its complicated sexuality, including dioecy, sex change and rare hermaphroditism. In this study, an integrated analysis of miRNA and mRNA expression profiles of oyster gonads at different gametogenesis stages was performed to explore their possibly regulatory patterns in sexual development of C. gigas. Based on small RNA sequencing, a total of 122 miRNAs were identified, including 53 conserved and 69 novel miRNAs. Six miRNAs exhibited sex-specific pattern that expressed exclusively from either male or female gonads with increasing abundance throughout the gonadal development. Moreover, 287 putative targets of the six sex-biased miRNAs were predicted by combining previous transcriptome data. GO and KEGG enrichment analysis of target genes revealed that these sex-biased miRNAs might participate in the regulation of gametogenesis. Further, potential miRNA-mRNA interactions based on six sex-biased miRNAs were collected. The homolog of a key participator in testis development, meiotic recombination protein SPO11 in C. gigas (CgSPO11), was predicted as a target of female-biased miRNA lgi-miR-96a. This negative miRNA-mRNA expression relationship was validated in additional samples by quantitative real-time PCR (qRT-PCR) and the regulation of CgSPO11 by lgi-miR-96a was preliminarily confirmed by a dual-luciferase reporter assay. This study provides information regarding the gonad miRNAs of C. gigas and leads to the discovery of miRNA-mRNA interactions in gonad development, which will facilitate further investigations in molecular mechanisms of bivalve sex differentiation. • A total of 122 miRNAs were identified during the Pacific oyster gonad development. • Six sex-biased miRNAs might participate in the regulation of oyster gametogenesis. • The CgSPO11 gene is a target of lgi-miR-96a. [ABSTRACT FROM AUTHOR]

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription–PCR (RT–PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses. [ABSTRACT FROM PUBLISHER]

Bicyclol is a synthetic antihepatitis drug with antioxidative property. This study was designed to investigate the effects of bicyclol on the activity, gene and protein expressions of hepatic microsomal cytochrome P450s (CYPs) during hepatic ischemia and reperfusion (I/R) in rats. The rats were subjected to 90 min of hepatic ischemia followed by reperfusion for 3 and 24 h. Bicyclol (300 mg/kg) was orally administered three times before hepatic ischemia in rats. Liver injury was evaluated by biochemical examinations. Hepatic microsomal malondialdehyde (MDA) was measured spectrophotometrically. Total hepatic CYP content and activities of four CYP isozymes were evaluated with a differential spectrophotometer and liquid chromatography-mass spectrometry analysis. The gene and protein expressions of four CYP isozymes were determined by reverse transcriptional-polymerase chain reaction and Western blotting assay. As a result, bicyclol significantly inhibited the elevation of serum alanine aminotransferase and hepatic microsomal MDA and prevented the decrease of total hepatic CYP content in I/R rats. In addition, bicyclol markedly attenuated the decrease of 2C6, 2C11, 3A1/2 activity and reduction of mRNA or protein expression of 2C6 and 3A in I/R rats. As for CYP2E1, bicyclol further aggravated the decrease of protein expression in I/R rats. These results suggested that bicyclol may ameliorate the abnormalities in the activity and expression of certain CYP isoforms during I/R, and this protective effect was likely due to its antioxidative and hepatoprotective properties. [ABSTRACT FROM AUTHOR]

OBJECTIVE -- A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve. RESEARCH DESIGN AND METHODS -- Microarray data for 24-week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further analyzed to identify regulated biological processes and pathways. Expression profile clustering was performed to identify coexpressed DEGs. A set of coexpressed lipid metabolism genes was used for promoter sequence analysis. RESULTS -- Gene expression changes are consistent with structural changes of axonal degeneration. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, peroxisome proliferator-activated receptor signaling, apoptosis, and axon guidance. Promoter sequences of lipid metabolism-related genes exhibit evidence of coregulation of lipid metabolism and nervous system development genes. CONCLUSIONS -- Our data support existing hypotheses regarding hyperglycemia-mediated nerve damage in DN. Moreover, our analyses revealed a possible coregulation mechanism connecting hyperlipidemia and axonal degeneration. [ABSTRACT FROM AUTHOR]

The mechanism of homocysteine-induced cell proliferation in human vascular smooth muscle cells (SMCs) remains unclear. We investigated the molecular mechanisms by which homocysteine affects the expression of cyclins A and D1 in human umbilical artery SMCs (HUASMCs). Homocysteine treatment induced proliferation of HUASMCs and increased the expression levels of cyclins A and D1. Knocking down either cyclin A or cyclin D1 by small interfering RNA (siRNA) inhibited homocysteine-induced cell proliferation. Furthermore, treatment with extracellular signal-related kinase (ERK) inhibitor (PD98059) and dominant negative Ras (RasN17) abolished homocysteine-induced cyclin A expression; and treatment with phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin) attenuated the homocysteine-induced cyclin D1 expression. Homocysteine also induced transient phosphorylation of ERK, Akt, and p70 ribosomal S6 kinase (p70S6K). Neutralizing antibody and siRNA for β1 integrin blocked cell proliferation, expression of cyclins A and D1, and phosphorylation of ERK and Akt. In conclusion, homocysteine-induced differential activation of Ras/ERK and PI3K/Akt/p70S6K signaling pathways and consequent expression of cyclins A and D1 are dependent on β1 integrin. Homocysteine may accelerate progression of atherosclerotic lesions by promoting SMC proliferation. J. Cell. Physiol. 226: 1017-1026, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Diabetic Retinopathy (DR) is one of the most common complications of diabetes and a major cause of blindness worldwide. We studied the transcriptome of the diabetic retina using Series Analysis of Gene Expression (SAGE) technology and observed a 45.6% reduction in transcript levels of glutamine synthetase (GS) in streptozotocin-induced diabetic rats compared with normal rats. RT-PCR and colorimetric enzyme activity assays revealed significant differences in GS mRNA expression and enzyme activity as early as the first month of diabetes development, with a progressive decrease in GS mRNA level and enzyme activity over a 12-month period. Northern blot analysis indicated a linear correlation between the reduction in GS expression and the time course of diabetic retinopathy (r = 0.802, p < 0.0001), which was validated by real-time RT-PCR (r = 0.731, p < 0.001). Our results implicate GS as a possible biomarker for evaluating the severity of developed diabetic retinopathy over the time course of diabetes progression. [Copyright &y& Elsevier]

Paramyosin is a key component of thick filaments in invertebrate muscles. In this study, we isolated the full length cDNA of paramyosin from Pacific oyster (Crassostrea gigas) , and determined its pattern of expression during myogenesis. The full length paramyosin (CgPM) cDNA contains an open reading frame (ORF) of 2586 bp encoding a 861-amino acid protein. Sequence analysis revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. These data indicate that CgPM is strongly expressed during larval myogenesis in C. gigas , which provides the basis for further functional studies of paramyosin in oyster to better understand the molecular and cellular mechanisms of muscle formation in mollusks. [Display omitted] • Here, the sequence characteristics and expression profiles of paramyosin in Crassostrea gigas (CgPM) were investigated. The main results are as follows: • Sequence analysis of CgPM revealed an assembly competence domain (ACD) and a heptad repeat (d-e-f-g-a-b-c) with 28-residue repeat zones in the CgPM primary structure, a characteristic of coiled-coil protein. • Quantitative analysis of CgPM expression revealed a sharp increase in trochophore stage, and peaked at the D-shaped stage. • Strong CgPM expression was found in smooth adductor muscle, followed by striated adductor muscle and mantle tissue. • Whole-mount in situ hybridization (WISH) showed a restricted pattern of CgPM expression in adductor muscle, larval velum retractor and foot muscles at the umbo and eyed larval stages. [ABSTRACT FROM AUTHOR]

Background: Super-enhancers (SEs) play a crucial role in cancer, which is often associate with activated oncogenes. However, little is known about how SEs facilitate tumour suppression. Individuals with Down syndrome exhibit a remarkably reduced incidence of breast cancer (BC), moving the search for tumor suppressor genes on human chromosome 21 (HSA21). In this study, we aim to identify and explore potential mechanisms by which SEs are established for tumor suppressor RCAN1.4 on HSA21 in BC. Methods: In silico analysis and immunohistochemical staining were used to assess the expression and clinical relevance of RCAN1.4 and RUNX3 in BC. Function experiments were performed to evaluate the effects of RCAN1.4 on the malignancy of breast carcinoma in vitro and in vivo. ChIP-seq data analysis, ChIP-qPCR, double-CRISPR genome editing, and luciferase reporter assay were utilized to confirm RUNX3 was involved in regulating RCAN1.4-associated SE in BC. The clinical value of co-expression of RCAN1.4 and RUNX3 was evaluated in BC patients. Results: Here, we characterized RCAN1.4 as a potential tumour suppressor in BC. RCAN1.4 loss promoted tumour metastasis to bone and brain, and its overexpression inhibited tumour growth by blocking the calcineurin-NFATc1 pathway. Unexpectedly, we found RCAN1.4 expression was driven by a ~ 23 kb-long SE. RCAN1.4-SEdistal was sensitive to BRD4 inhibition, and its deletion decreased RCAN1.4 expression by over 90% and induced the malignant phenotype of BC cells. We also discovered that the binding sites in the SE region of RCAN1.4 were enriched for consensus sequences of transcription factor RUNX3. Knockdown of RUNX3 repressed the luciferase activity and also decreased H3K27ac enrichment binding at the SE region of RCAN1.4. Furthermore, abnormal SE-driven RCAN1.4 expression mediated by RUNX3 loss could be physiologically significant and clinically relevant in BC patients. Notably, we established a prognostic model based on RCAN1.4 and RUNX3 co-expression that effectively predicted the overall survival in BC patients. Conclusions: These findings reveal an important role of SEs in facilitating tumour suppression in BC. Considering that the combination of low RCAN1.4 and low RUNX3 expression has worse prognosis, RUNX3-RCAN1.4 axis maybe a novel prognostic biomarker and therapeutic target for BC patients. [ABSTRACT FROM AUTHOR]

A series of studies have confirmed that DNA methylation disorder (5mC/5hmC) is closely related to the occurrence and development of some diseases, such as Alzheimer's disease (AD). This study aims at discovering natural compounds that could adjust and control 5-hydroxymethylcytosine (5hmC) levels and improve Alzheimer's disease (AD) neuronal status. Cordycepin and cordycepic acid were selected as research materials, with resveratrol as positive control. The results of Dot Blot indicated that cordycepin, cordycepic acid, and resveratrol significantly increased the expression level of 5hmC. Combined with qPCR results, it was revealed that cordycepin increased the expression of ten-eleven translocation (TETs) mRNA compared with the abovementioned cordycepic acid and resveratrol. Besides, cordycepin dramatically reduced the transcription level of Apolipoprotein E (ApoE), suggesting that cordycepin might hinder the formation of NFTs (neurofibrillary tangles) and the accumulation of amyloid β-protein (Aβ) in the brain by reducing the expression of ApoE, resulting in affecting the progression of AD. Meanwhile, the immunofluorescence (IF) staining results demonstrated that the percentage of differentiation of SHSY-5Y cells reasonably increased after the treatment of cordycepin and cordycepic acid. Simultaneously, the length of axons and the number of dendritic branches in mouse primary neurons were substantially increased by cordycepin. The screening results illustrated that cordycepin had a positive influence on the level of 5hmC and the morphology of neurons, and most of the effects were better compared to the positive control (resveratrol). It indicated that cordycepin delayed the progression of neurodegenerative diseases such as AD. However, the specific mechanism of action still needs to be further investigated. Our research provided a foundation for further discussion about the influence of cordycepin on AD and a new idea for the pathological study of related diseases. [ABSTRACT FROM AUTHOR]

Introduction: Severe aplastic anemia (SAA) is a disease characterized by severe pancytopenia and hematopoietic failure of bone marrow. Natural killer (NK) cells are a class of large granular lymphocytes that perform killing and immunomodulatory functions. Our previous study demonstrated that NK cells played the "protective" role in SAA, which is weakened. However, the mechanism remains unclear. Methods: Peripheral blood NK cells from SAA patients and normal controls were sorted and total proteins were extracted. Then, mass spectrometry was performed to screen differentially expressed proteins (DEPs). Results: Significant differences in the expression levels of 93 proteins were observed in NK cells of SAA patients compared with normal controls. Among them, 48 were upregulated proteins, including histone H1.2, histone H1.3, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), and interferon regulatory factor 1 (IRF‐1), and 45 were downregulated proteins, including actin‐related complex (ARP2/3), histone H3, histone H4, phosphoglycerate kinase 1 (PGK1), talin‐1. Gene Ontology (GO) function indicated that the DEPs most involved were vesicle‐mediated transport, innate immune response, and DNA binding. KEGG analysis showed 3 upregulated and 12 downregulated pathways, in which cell endocytosis and FC‐γ receptor‐mediated phagocytosis were most closely related to NK cell functions. Conclusion: Our study is the first analysis of proteomic profile in NK cells in SAA and found many DEPs involving in dysfunction of NK cells, which provides potential targets for deeper research of inadequate immunomodulation. [ABSTRACT FROM AUTHOR]

The underlying mechanisms of macrophage polarization have been detected by genome-wide transcriptome analysis in a variety of mammals. However, the transcriptome profile of rat genes in bone marrow-derived macrophages (BMM) at different activation statuses has not been reported. Therefore, we performed RNA-Sequencing to identify gene expression signatures of rat BMM polarized in vitro with different stimuli. The differentially expressed genes (DEGs) among unactivated (M0), classically activated pro-inflammatory (M1), and alternatively activated anti-inflammatory macrophages (M2) were analyzed by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. In this study, not only we have identified the changes of global gene expression in rat M0, M1 and M2, but we have also made clear systematically the key genes and signaling pathways in the differentiation process of M0 to M1 and M2. These will provide a foundation for future researches of macrophage polarization. • The transcriptome profile of rat genes in bone marrow-derived macrophages at different activation statuses is investigated. • The global gene expression in rat M0, M1 and M2 has been described. • The key genes and signaling pathways in the differentiation process of M0 to M1 and M2 have been systematically determined. • Our results will provide a basis for future researches of macrophage polarization. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Chrysanthemum indicum Linne (C. indicum), a healthy food and folk medicine in China for thousands of years, has been reported to exert heat-clearing and detoxifying effects and extensively applied to treat various symptoms such as inflammation diseases, hepatitis and headache. Aim of this study The purpose of the present study was to investigate the protective effect of the supercritical carbon dioxide fluid extract from flowers and buds of C. indicum (CI SCFE) on D -galactose-induced brain and liver damage during aging process and to illuminate the underlying mechanisms. Materials and methods Mice were orally administrated with CI SCFE (100, 150 and 300 mg/kg) after injection with D -galactose. 24 h after the last administration, the blood samples, whole brain and liver tissues were collected for biochemical analysis, histological examination and western blot analysis. The body weight, spleen and thymus indexes, alanine transaminase (ALT), aspartate transaminase (AST), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) in brain and liver, interleukin-1β (IL-1β), interleukin-6 (IL-6), and necrosis factor-α (TNF-α) were detected. Besides, the expressions of Bax, Bcl-2 and cleaved caspase-3 were determined by western blot assay. Results The results indicated that CI SCFE effectively increased the suppressed body weight, attenuated the decline of thymus and spleen indexes, and reduced the elevated levels of ALT and AST induced by D -gal. Furthermore, CI SCFE might notably alleviate D -gal-induced abnormal alterations in structure and function of brain and liver dose-dependently via renewing normal antioxidant enzymes activities (SOD, CAT, GSH-Px), reducing MDA accumulation, decreasing inflammatory cytokines productions (IL-1β, IL-6, TNF-α), as well as attenuating the increase of Bax/Bcl-2 ratio and cleaved caspase-3 activation in the liver and brain. Conclusions Taken together, our present results suggested that CI SCFE treatment could effectively mitigate the D -gal-induced hepatic and cerebral injury, and the underlying mechanism might be tightly related to the decreased oxidative stress, inflammation and apoptosis, indicating CI SCFE might be an alternative and promising agent for the treatment of aging and age-associated brain and liver diseases. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most common malignancy tumors with insidious onset, rapid development and metastasis, and poor prognosis. Therefore, it is necessary to understand molecular mechanisms of HCC and identify clinically useful biomarkers for it. This study aimed to investigate the role of retinoblastoma binding protein 5 (RBBP5) in HCC. The expression level of RBBP5 was examined by immunohistochemistry and western blot. The effect of RBBP5 on cell cycle, proliferation, apoptosis, and drug sensitivity was analyzed. RBBP5 was significantly upregulated in HCC tissues and cells. High RBBP5 expression was significantly associated with elevated level of AFP, advanced TNM stage, high Ki-67 expression, larger tumor size, and poor prognosis. Knockdown of RBBP5 significantly inhibited proliferation of HCC cells through cell cycle arrest. In addition, inhibition of RBBP5 increased the sensitivity of HCC cells to doxorubicin. In conclusion, our findings suggest that RBBP5 plays an important role in the progression of HCC and may serve as a novel biomarker and potential therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

To systematically unveil transcription factors (TFs) that are critical to lung carcinogenesis, here we conducted a genome-wide lethality screening in non-small cell lung cancer (NSCLC) cells and reported that among the 1530 TFs tested, 21 genes were required for NSCLC cell proliferation and were negatively or positively associated with overall survival (OS) of patients with NSCLC. These included 11 potential tumor suppressing genes (AFF3, AhR, AR, CBFA2T3, CHD4, KANK2, NR3C2, PTEN, PRDM16, RB1, and STK11) and 10 potential oncogenic TFs (BARX1, DLX6, ELF3, EN1, ETV1, FOXE1, HOXB7, IRX4, IRX5, and SALL1). The expression levels of IRX5 were positively associated with OS of smoker and inversely associated with OS of non-smoker patients with lung adenocarcinoma. We showed that tobacco carcinogen benzo(a)pyrene (BaP) induced upregulation of IRX5 in lung epithelial cells, and Cyclin D1 was a downstream target of IRX5. Furthermore, silencing of IRX5 by lentivirus mediated transfection of short hairpin RNA significantly inhibited tumor growth in nude mice. These results indicate that tobacco smoke can modulate TFs to facilitate lung carcinogenesis, and inhibition of IRX5 may have therapeutic potentials in NSCLCs. [ABSTRACT FROM AUTHOR]

Icariin (ICA) and icaritin (ICT) exhibit many pharmacological functions including anti-osteoporosis, anti-cardiovascular, and anti-cancer activities; however, there are few comprehensive studies that track the detailed effects on UVB-induced photoaging. The recovery effects of ICA and ICT were investigated in UVB-irradiated human keratinocytes (HaCaTs). The results indicated that ICT and ICA showed strong radical scavenging activity, and the reactive oxygen species (ROS) scavenging activity of ICT was superior. UVB-induced matrix metalloproteinase-1 (MMP-1) expression was blocked by ICA via the inhibition of mitogen-activated protein kinase/activator protein 1 (MAPK/AP-1), which directly reduced extracellular matrix (ECM) degradation. ICT activated nuclear factor erythroid 2 related factor 2 (Nrf2) to improve the anti-oxidative stress capacity and suppress nuclear factor-κB (NF-κB) activation, decreasing vascular endothelial growth factor (VEGF) protein, and inflammatory cytokines induced ECM degrading enzyme secretion. Moreover, ICT was more advantageous to improve transforming growth factor beta 1 (TGF-β1) and procollagen type I expression than ICA, promoting the synthesis of collagen. Therefore, ICA and ICT have potential to treat UVB-induced oxidative stress, inflammation and photoaging, and will be posited as a novel strategy to alleviate photodamage. [ABSTRACT FROM AUTHOR]

We investigated the beneficial effects and underlying mechanisms of Zhuanggu Guanjie (ZGGJ) pill in osteoporosis in vitro and in vivo. Bone marrow macrophages from 4–6-week-old mice were cultured in the presence of macrophage colony-stimulating factor (15 ng/mL) and receptor activator of nuclear factor-κB ligand (30 ng/mL). Osteoclast differentiation was determined by quantification of tartrate-resistant acid phosphatase activity. Gelatin zymography was used to detect the activity of matrix metalloproteinases in osteoclasts. Ovariectomized rats were administered orally with estradiol valerate or ZGGJ for 8 weeks. Blood was collected to measure serum indices. Tibiae were harvested to carry out bone microcomputed tomography scanning, histomorphological analysis, and bone strength determination. ZGGJ inhibited tartrate-resistant acid phosphatase activity, matrix metalloproteinase 9 expression, and bone resorption in vitro. At doses of 0.55, 1.1, and 2.2 g/kg, ZGGJ exerted significant osteoprotective effects including inhibition of bone turnover markers and improved tibia bone strength in ovariectomized rats. Microcomputed tomographic analysis showed that ZGGJ improved the trabecular architecture with increased connectivity density and trabecular thickness and decreased trabecular spacing. These results revealed that ZGGJ prevents bone loss induced by ovariectomy in rats and that inhibition of bone resorption is involved in the bone-protective effects of ZGGJ. [ABSTRACT FROM AUTHOR]

MiRNAs regulated most genes expression, which were proved important in various tumors. In this study, we want to investigate miR-101 effect and molecular mechanism on pancreatic cancer (PC), the research about this was blank now. RT-PCR analysis showed that miR-101 expression was declined in PC. MTT assay found that miR-101 mimic suppressed cell viability, while suppressing miR-101 facilitated cell proliferation. Transwell assay showed that miR-101 mimic inhibited cell invasion, but promoted cell invasion by miR-101 inhibitor. With TargetScanHuman's help, we verified STMN1 as a specific target of miR-101 and luciferase reporter assay was carried out to further confirm this discovery. STMN1 expression was reduced by miR-101 mimic and increased by miR-101 inhibitor. We next found that STMN1 was elevated in PC and its expression was negatively correlated with miR-101 expression. Furthermore, STMN1 siRNA curbed cell proliferation and invasion, which was opposite to miR-101 inhibitor effect on PC progression and STMN1 siRNA attenuated miR-101 inhibitor effect on cell proliferation and invasion. In conclusion, miR-101 inhibited PC cell proliferation and invasion via regulating STMN1, which provided a potential therapeutic for PC patients. [ABSTRACT FROM AUTHOR]

Laryngeal squamous cell carcinoma (LSCC) is the most lethal cancer in head and neck tumors. Although hematopoietic cell kinase (HCK) has been proven to be an oncogene in several solid tumors, its roles in LSCC remain obscure. This is the first study to evaluate the clinical value of HCK in LSCC, with the aim of exploring its expression status and potential molecular mechanisms underlying LSCC. LSCC tissue-derived gene chips and RNA-seq data were collected for a quantitive integration of HCK mRNA expression level. To confirm the protein expression level of HCK, a total of 82 LSCC tissue specimens and 56 non-tumor laryngeal epithelial controls were collected for in-house tissue microarrays and immunohistochemical staining. Kaplan-Meier curves were generated to determine the ability of HCK in predicting overall survival, progress-free survival, and disease-free survival of LSCC patients. LSCC overexpressed genes and HCK co-expressed genes were intersected to preliminarily explore the enriched signaling pathways of HCK. It was noticed that HCK mRNA was markedly overexpressed in 323 LSCC tissues compared with 196 non-LSCC controls (standardized mean difference = 0.81, p < 0.0001). Upregulated HCK mRNA displayed a moderate discriminatory ability between LSCC tissues and non-tumor laryngeal epithelial controls (area under the curve = 0.78, sensitivity = 0.76, specificity = 0.68). The higher expression level of HCK mRNA could predict worse overall survival and disease-free survival for LSCC patients (p = 0.041 and p = 0.013). Lastly, upregulated co-expression genes of HCK were significantly enriched in leukocyte cell-cell adhesion, secretory granule membrane, and extracellular matrix structural constituent. Immune-related pathways were the predominantly activated signals, such as cytokine-cytokine receptor interaction, Th17 cell differentiation, and Toll-like receptor signaling pathway. In conclusion, HCK was upregulated in LSCC tissues and could be utilized as a risk predictor. HCK may promote the development of LSCC by disturbing immune signaling pathways. [ABSTRACT FROM AUTHOR]

Cardiac ankyrin repeat protein (CARP) is a nuclear transcriptional co-factor that has additional functions in the myoplasm as a component of the muscle sarcomere. Previous studies have demonstrated increased expression of CARP in cardiovascular diseases, however, its role in cardiomyocyte apoptosis is unclear and controversial. In the present study, we investigated possible roles of CARP in hypoxia/reoxygenation (H/R) -induced cardiomyocyte apoptosis and the underlying mechanisms. Neonatal mouse ventricular cardiomyocytes were isolated and infected with adenovirus encoding Flag-tagged CARP (Ad-CARP) and lentivirus encoding CARP targeted shRNA (sh-CARP), respectively. Cardiomyocyte apoptosis induced by exposure to H/R conditions was evaluated by TUNEL staining and western blot analysis of cleaved caspase-3. The results showed that H/R-induced apoptosis was significantly decreased in Ad-CARP cardiomyocytes and increased in sh-CARP cardiomyocytes, suggesting a protective anti-apoptosis role for CARP. Interestingly, over-expressed CARP was mainly distributed in the nucleus, consistent with its role in regulating transcriptional activity. qPCR analysis showed that Bcl-2 transcripts were significantly increased in Ad-CARP cardiomyocytes. ChIP and co-IP assays confirmed the binding of CARP to the Bcl-2 promoter through interaction with transcription factor GATA4. Collectively, our results suggest that CARP can protect against H/R induced cardiomyocyte apoptosis, possibly through increasing anti-apoptosis Bcl-2 gene expression. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning and ischemic postconditioning (IPostC) represent promising strategies to reduce ischemia-reperfusion (I/R) injury and attenuate the lethal ischemic damage following stroke. However, the mechanism underlying this attenuation remains to be elucidated. It was hypothesized that alterations in microRNA (miRNA) expression in the cerebral cortex and hippocampus of mice following I/R is associated with the functional improvement induced by IPostC. Behavioral changes were assessed in a mouse model of I/R in the absence or presence of IPostC, followed by microarray analyses to investigate the expressional alterations of miRNAs in the cerebral cortex and hippocampus of mice. The results of the present study revealed that IPostC abrogated the neurological impairment and hippocampus-associated cognitive deficits induced by I/R, and upregulated or downregulated the expression levels of numerous miRNAs. Furthermore, the upregulation of miR-19a, and the downregulation of miR-1, let-7f and miR-124 expression levels following IPostC was confirmed utilizing reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that alterations in miRNA expression in the cerebral cortex and hippocampus of mice following I/R was associated with the neuroprotection induced by IPostC. [ABSTRACT FROM AUTHOR]

Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which depends on the orchestrated accessibility of regulatory proteins to promoters and other cis -regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the developmental process. To understand the regulatory mechanism and functional elements modulating morphological and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites (DHSs) from the fruit tissues of the tomato cultivar “Moneymaker” at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening. [ABSTRACT FROM AUTHOR]

Aim Hyperlipidemia is a disease with abnormally elevated levels of lipids/lipoproteins in the blood, and it is regarded as an important risk factor for cardiovascular and cerebrovascular diseases. Statins have been found to prevent vascular diseases by reducing low-density lipoprotein cholesterol and regulation of immune responses. Here, we aim to study the expression change of immune-related microRNA and genes in older patients with hyperlipidemia after treatment with simvastatin. Methods A total of 25 older male patients with hyperlipidemia were included in the study and received simvastatin treatment (20 mg/day). Clinical characteristics of these patients were examined, including lipoprotein cholesterol, high-sensitivity C-reactive protein, blood routine and biochemical characters. We tested mi R-146a, interleukin-1-receptor-associated kinase 1, tumor necrosis factor-receptor-associated factor 6 and cyclooxygenase-2 level by real-time polymerase chain reaction, and expressions of advanced glycation end-products, p53 and p21 were analyzed by enzyme-linked immunosorbent assay. Results Simvastatin treatment effectively reduced total cholesterol and low-density lipoprotein cholesterol, but had little effect on high-density lipoprotein cholesterol. High-sensitivity C-reactive protein was slightly reduced. Expression of cyclooxygenase-2 and advanced glycation end-products were significantly reduced. Furthermore, simvastatin effectively reduced the expression of p53 and p21. Significantly downregulated mi R-146a, and an obvious reduction of interleukin-1-receptor-associated kinase 1were also detected, whereas tumor necrosis factor-receptor-associated factor 6 remained unchanged. Besides, there was a significant reduction of alanine transaminase, aspertate aminotransferase, alkaline phosphatase and lactate dehydrogenase. Conclusion Simvastatin treatment could inhibit inflammation and senescence-associated genes in older patients with hyperlipidemia, suggesting its application in inflammatory and age-related diseases. Geriatr Gerontol Int 2015; ●●: ●●-●●. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis. [ABSTRACT FROM AUTHOR]

Background: Prenatal dexamethasone exposure has been reported to increase allergy potential in childhood possibly by interference with normal immunological development in utero. This study investigated the effects of prenatal dexamethasone on T helper cell immune responses in a rat model. Methods: Pregnant rats received either dexamethasone 0.1 mg/kg/day or normal saline from gestational day 14–21. Off-springs were cared for by their biological mother, or cross-fostered by the opposing group. Spleen and blood samples were collected at post-natal day 7 and 120 and tested for mRNA expression and plasma cytokine levels of Th1/Th2/Th17 immune response. Results: Both Th1 (T-bet) and Th2 (GATA-3) mRNA expression were shown to have a significant increase in the prenatal dexamethasone exposure group at day 120 (p<0.05). The plasma levels for Th1 (IFNγ and IL-2) and Th2 (IL-4, IL-5, IL-13) were found to have no significant differences between the two group (p>0.05). The mRNA expression of Th17 (RORγt) showed a significant decrease at post-natal day 120 as well as the plasma level of IL-17A at day 7 (11.21±1.67 vs. 6.23±1.06 pg/ml, p = 0.02). Cross-fostering by a dexamethasone exposed mother resulted in a significant increase in Th1/Th2 mRNA expression (p<0.05) and decrease of Th17. Conclusions: Prenatal dexamethasone exposure increased Th1, Th2 and decreased Th17 expression. Cross-fostering by a dexamethasone exposed mother results in more prominent increase of Th1 and Th2 expression. [ABSTRACT FROM AUTHOR]

Background: The β-catenin is an important effector in WNT/β-catenin signaling pathway, which exerts a crucial role in the development and progression of hepatocellular carcinoma (HCC). Some researchers have suggested that the overexpression of β-catenin in cytoplasm and/or nucleus was closely correlated to metastasis, poor differentiation and malignant phenotype of HCC while some other researchers hold opposite point. So far, no consensus was obtained on the prognostic and clinicopathological significance of cytoplasmic/nuclear β-catenin overexpression for HCCs. Methods: Systematic strategies were applied to search eligible studies in all available databases. Subgroup analyses, sensitivity analyses and multivariate analysis were performed. In this meta-analysis, we utilized either fixed- or random-effects model to calculate the pooled odds ratios (OR) and its 95% confidence intervals (CI). Results: A total of 22 studies containing 2334 cases were enrolled in this meta-analysis. Pooled data suggested that accumulation of β-catenin in cytoplasm and/or nucleus significantly correlated with poor 1-, 3- and 5-year OS and RFS. Moreover, nuclear accumulation combined with cytoplasmic accumulation of β-catenin tended to be associated with dismal metastasis and vascular invasion while cytoplasmic or nuclear expression alone showed no significant effect. Besides, no significant association was observed between cytoplasmic and/or nuclear β-catenin expression and poor differentiation grade, advanced TNM stage, liver cirrhosis, tumor size, tumor encapsulation, AFP and etiologies. Additional subgroup analysis by origin suggested that the prognostic value and clinicopathological significance of cytoplasmic and/or nuclear β-catenin expression was more validated in Asian population. Multivariate analyses of factors showed that cytoplasmic and/or nuclear β-catenin expression, as well as TNM stage, metastasis and tumor size, was an independent risk factors for OS and RFS. Conclusions: Cytoplasmic and/or nuclear accumulation of β-catenin, as an independent prognostic factor, significantly associated with poor prognosis and deeper invasion of HCC, and could serve as a valuable prognostic predictor for HCC. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of pentylenetetrazol (PTZ) and nuclear factor κ B (NF-κB) decoy oligodeoxynucleotides (ODNs) on p38 expression in neuron-like PC12 cells. In addition, the role of NF-κB activation in the pathogenesis of epilepsy was explored. p38 expression levels in control and PTZ-treated neuron-like PC12 cells were examined using western blotting. NF-κB decoy ODNs were transfected into the neuron-like PC12 cells using Lipofectamine 2000. NF-κB activation was investigated by confocal laser scanning microscopy (CLSM), and p38 expression levels were assessed using western blotting prior to and following transfection of decoy ODNs. Western blot analysis revealed that p38 levels in PTZ-treated neuron-like PC12 cells were significantly higher than those in control cells. CLSM demonstrated that the decoy ODNs inhibited NF-κB activation in neuron-like PC12 cells, and western blotting indicated that the decoy ODNs did not reduce p38 levels. The results of this study indicate that PTZ enhances p38 expression levels and activates NF-κB in PC12 cells. However, NF-κB does not modulate p38 expression levels. [ABSTRACT FROM AUTHOR]

Simple Summary: Quantitative real-time fluorescent polymerase chain reaction (qRT-PCR) is a momentous tool for calculating the expression levels of targeted genes across various experimental conditions. The selection and evaluation of stable reference genes for qRT-PCR analysis is an essential precondition for reliable expression assessment. Phthorimaea operculella is one of the most serious Lepidopteran pests that attack potatoes around the world. In the present paper, a total of 10 commonly used reference genes, namely ACT, α-TUB, 18S, 28S, GAPDH, EF1α, RPL4, RPL13, RPL27 and SOD, were selected and validated for suitability under three treatments (developmental stages, tissues/organs and temperatures) using five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). These results indicated that EF1α and RPL13 were the best suitable reference genes for diverse backgrounds. The relative transcript levels of the target gene chitin synthase A gene (PoChSA) were abundantly expressed in epidermal cells, and lowly transcribed in the midgut. Our findings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in P. operculella. Due to a lack of effective internal references, studies on functional genes in Phthorimaea operculella, a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (ACT), α-tubulin (α-TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), 18S and 28S ribosomal RNA (18S, 28S), ribosomal protein genes RPL4, RPL13 and RPL27 and superoxide dismutase (SOD) were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that RPL13, EF1α and RPL27 are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; EF1α and RPL13 are recommended to normalize the gene expression levels among diverse tissues. EF1α and RPL13 are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, EF1α and RPL13, we estimated the tissue-biased expression level of chitin synthase A gene (PoChSA). As expected, PoChSA was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of P. operculella. [ABSTRACT FROM AUTHOR]

Abstract: The tumor suppressor gene phosphatase and tensin homolog (PTEN) is essential in inhibiting tumor growth and metastasis. However, the mechanism by which PTEN restricts gastric cancer progression and metastasis remains largely elusive. Here we demonstrated that PTEN overexpression or knockdown in gastric cancer cells led to the downregulation or upregulation of focal adhesion kinase (FAK), and decreased or increased cell invasion, respectively. Moreover, FAK overexpression could rescue the inhibition of cell invasion by PTEN. These results were further confirmed in orthotropic gastric cancer nude mice model. In addition, in human gastric cancer tissues, PTEN protein level was conversely correlated with FAK protein level. Mechanistically, we found that PTEN inhibited PI3K/NF-κB pathway and inhibited the DNA binding of NF-κB on FAK promoter. Taken together, our data reveal a novel mechanism that PTEN inhibits the growth and invasion of gastric cancer via the downregulation of FAK expression and suggest that exploiting PTEN/PI3K/NF-κB/FAK axis is a promising approach to treat gastric cancer metastasis. [Copyright &y& Elsevier]

Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2–CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development. [ABSTRACT FROM PUBLISHER]

Adiponectin and adiponectin receptors (AdipoR1/2) are expressed in various tissues and are involved in the regulation of multiple functions such as energy metabolism and inflammatory responses. However, the effect of adiponectin and AdipoRs in submandibular glands has not been fully evaluated. In the present study, we found that mRNA and protein of both adiponectin and AdipoR1/2 were expressed in rat submandibular glands and in the SMG-C6 cell line, as evidenced by RT-PCR and Western blot analysis. Immunofluorescence staining showed that adiponectin was diffused in the cytoplasm, while AdipoR1/2 was concentrated in the membrane of acinar cells. Saliva flow was significantly increased by full length adiponectin (fAd) or globular adiponectin (gAd) perfusion in isolated rat submandibular glands. 5-Aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR), an adenosine monophosphate activated protein kinase (AMPK) activator, also increased saliva secretion. fAd, gAd, and AICAR all increased the average width of apical tight junctions in perfused submandibular glands, and decreased transepithelial electrical resistance (TER) in SMG-C6 cells, suggesting that adiponectin promoted secretion by modulating paracellular permeability. fAd and gAd increased p-AMPK levels, while AraA, an AMPK antagonist, abolished fAd- and gAd-induced changes in secretion, tight junction ultrastructure, and TER. Moreover, both AdipoR1 and AdipoR2 were required for fAd- or gAd-induced p-AMPK and TER responses, suggesting from their inhibition following AdipoR1 or AdipoR2 knockdown, and co-knockdown of AdipoRs by RNA interference. Our results suggest that adiponectin functions as a promoter of salivary secretion in rat submandibular glands via activation of AdipoRs, AMPK, and paracellular permeability. [ABSTRACT FROM AUTHOR]

Bacterial chromosomal toxin-antitoxin (TA) systems have been proposed not only to play an important role in the stress response, but also to be associated with antibiotic resistance. Here, we identified the chromosomal HP0892-HP0893 TA proteins in the gastric pathogen, Helicobacter pylori, and structurally characterized their protein-protein interaction. Previously, HP0892 protein was suggested to be a putative TA toxin based on its structural similarity to other RelE family TA toxins. In this study, we demonstrated that HP0892 binds to HP0893 strongly with a stoichiometry of 1:1, and HP0892- HP0893 interaction occurs mainly between the N-terminal secondary structure elements of HP0892 and the C-terminal region of HP0893. HP0892 cleaved mRNA in vitro, preferentially at the 5' end of A or G, and the RNase activity of HP0892 was inhibited by HP0893. In addition, heterologous expression of HP0892 in Escherichia coli cells led to cell growth arrest, and the cell toxicity of HP0892 was neutralized by co-expression with HP0893. From these results and a structural comparison with other TA toxins, it is concluded that HP0892 is a toxin with intrinsic RNase activity and HP0893 is an antitoxin against HP0892 from a TA system of H. pylori. It has been known that hp0893 gene and another TA antitoxin gene, hp0895, of H. pylori, are both genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. Therefore, it is highly probable that TA systems of H. pylori are involved in virulence of H. pylori. [ABSTRACT FROM AUTHOR]

Background: Zinc finger, DHHC-type containing 2 (ZDHHC2), originally named as reduced expression associated with metastasis protein (REAM), has been proposed as a putative tumor/metastasis suppressor gene and is often aberrantly decreased in human cancers. However ZDHHC2 expression pattern and its clinical significance have not yet been investigated in gastric adenocarcinoma. Methodology/Principal Findings: Quantitative Real-Time PCR (qRT-PCR) and immunostaining were performed to detect ZDHHC2 expression in gastric adenocarcinoma, and then the correlation between ZDHHC2 expression and clinicpathologic parameters, and patient survival was analyzed. Compared to the adjacent normal tissues, ZDHHC2 expression was significantly reduced in gastric tumor tissues as shown by qRT-PCR and immunostaining. Low expression of ZDHHC2 was observed in 44.7% (211/472) of gastric adenocarcinoma patients, and was associated significantly with lymph node metastasis (p<0.001) and histological grade (p<0.001). Multivariate Cox regression analysis indicated that ZDHHC2 expression had a significant, independent predictive value for survival of gastric cancer patients (HR = 0.627, p = 0.001). Conclusions/Significance: Our data suggest that reduced ZDHHC2 expression is associated with lymph node metastasis and independently predicts an unfavorable prognosis in gastric adenocarcinoma patients. [ABSTRACT FROM AUTHOR]

Purpose: Recent reports have linked various autoimmune diseases to defective Fasmediated apoptosis or Fas expression. Here we aimed to determine whether Fas-mediated apoptosis is involved in the pathogenesis of myasthenia gravis (MG). Methods: The expression of Fas antigen in peripheral T cell subsets from 17 Chinese patients with MG and 13 healthy individuals was determined by flow cytometry, and its associations with clinical classification, thymus pathology, the concomitance with hyperthyroidism (HT) and corticosteroid treatment were investigated. Results: Compared with normal controls, a significantly up-regulated expression of Fas antigen was observed in the peripheral CD4+, CD4+CD8- and CD4-CD8- T cell subsets from patients with MG. Fas expression in CD4-CD8+ T cells of MG patients with normal thymus was significantly higher than that of patients with thymoma. Fas expressions in CD4+CD8+ T cells in MG patients with HT was significantly higher than controls and the ones without HT. Enhanced Fas expressions was found in CD4-CD8+ and CD4-CD8- T cells of MG patients with corticosteroid treatment, but no significant difference of Fas expression in peripheral T cells between patients with ocular MG (OMG) and general MG (GMG) was observed. Conclusion: Fas antigen may play a role in the pathogenesis of MG. It may be involved in the mechanisms of corticosteroid treatment, and with the occurrence of HT. OMG may represent a systemic disease, similar to that of GMG. [ABSTRACT FROM AUTHOR]

Abstract: Aim: To examine the in vitro and in vivo effects of a combined treatment of cis-d iamminedichloroplatinum(II) (cisplatin) with downregulation of Akt1 expression in gastric cancer cells. Materials and methods: Lentivirus-mediated RNA interference (RNAi) was used to silence the Akt1 gene. pGCSIL-Akt1 small hairpin RNA (shRNA) was stably transfected into gastric cancer cells (SGC7901 and BGC823). Next, the effects of Akt1 downregulation on the growth and apoptosis of SGC7901 (BGC823) cells in the presence or absence of cisplatin were investigated by real-time polymerase chain reaction (RT-PCR), Western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-d-iphenyltetrazolium bromide) assay, Hoechst assay, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). Finally, the effects of downregulation of Akt1 expression on the sensitivity of SGC7901 cells in a tumor xenograft model of cisplatin were also determined. Result: Akt1 silencing reduced gastric cancer proliferation and increased cell apoptosis both in vitro and in vivo. The chemosensitivity of SGC7901 (BGC823) cells to cisplatin increased significantly following the downregulation of Akt1 expression, which might be associated with the inactivation of the PI3K/Akt1 signaling pathway, followed by the induced expression of the pro-apoptotic protein Bax and a concomitant decrease of Bcl-2 expression. Conclusion: This study confirmed that downregulation of Akt1 reduced chemotherapy tolerance of gastric cancer cells to cisplatin treatment. Thus, Akt1 silencing and cisplatin appear to be an effective combination treatment strategy for gastric cancer. [Copyright &y& Elsevier]

Abstract: Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato. [Copyright &y& Elsevier]

Abstract: Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na+/H+ transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii, a pea shrub with high tolerance to salt, drought, and cold stresses. Transcripts of CkNHX1 were detected predominantly in roots, and were significantly induced by NaCl stress in stems. Transgenic yeast and Arabidopsis thaliana sos3-1 (Atsos3-1) mutant over-expressing CkNHX1 and its hydrophilic C terminus-truncated derivative, CkNHX1-ΔC, were generated and subjected to NaCl and LiCl stresses. Expression of CkNHX1 significantly enhanced the resistance to NaCl and LiCl stresses in yeast and Atsos3-1 mutant. Whereas, compared with expression of CkNHX1, the expression of CkNHX1-ΔC had much less effect on NaCl tolerance in Atsos3-1 and LiCl tolerance in yeast and Atsos3-1. All together, these results suggest that the predominant expression of CkNHX1 in roots might contribute to keep C. korshinskii adapting to the high salt condition in this plant’s living environment; CkNHX1 could recover the phenotype of Atsos3-1 mutant; and the hydrophilic C-terminal region of CkNHX1 should be required for Na+/H+ and Li+/H+ exchanging activity of CkNHX1. [Copyright &y& Elsevier]

Abstract: Objectives: Reduced numbers and activity of circulating progenitor cells are associated with aging and have been linked with coronary artery disease. To determine the impact of aging and atherosclerotic disease on the chemotaxic activity of bone marrow derived cells (BMCs), we examined CXCR4 surface expression on BMCs from aged and atherosclerotic mice. Methods: CXCR4 expression and cellular mobility were compared between BMCs of young (6-week old) ApoE null mice (ApoE−/−) and aged ApoE−/− mice that had been fed with a high-fat, high-cholesterol diet for 6-months. Results: Age and atherosclerosis correlated with significantly lower surface expression of CXCR4 that was less inducible by calcium. The impaired calcium response was associated with defective calcium influx and was partially recovered by treatment with the calcium ionophore ionomycin. ApoE−/− mice fed high fat diet for 6-months had defective CXCR4 expression and SDF-1 regulation that is equivalent to that of 24-month old wild type mice. BMCs from aged, atherogenic ApoE−/− mice also displayed defective homing to SDF-1, and the animals had lower serum and bone marrow levels of SDF-1. Conclusion: Evolution of atherosclerosis in ApoE−/− mice is paralleled by progressive loss of mobility of BMCs with reductions of CXCR4 expression, and reduced levels of SDF-1 in both serum and bone marrow. These changes mute the homing capability of BMCs and may contribute to the progression of atherosclerosis in this model. [Copyright &y& Elsevier]

Abstract: We previously reported that exogenous H2S played roles in protection of respiratory centers against hypoxic injury in medullary slices of neonatal rats. The protective action of endogenous H2S and its relation to antioxidation and down-regulation of c-fos mRNA were investigated in the present study. Perfusion of the slices with l-cysteine (Cys), substrate of cystathionine β-synthase (CBS, H2S synthase), could increase frequency of rhythmic respiratory discharge of the hypoglossal rootlets and prevent respiratory suppression induced by hypoxia, whereas perfusion with hydroxylamine (NH2OH, inhibitor of CBS) could postpone recovery of respiration from hypoxic inhibition. NH2OH also significantly enhanced hypoxia-induced increase in malondialdehyde (MDA) content of the slices. The hypoxia-induced up-regulation of c-fos mRNA could be markedly antagonized by S-adenosyl-l-methionine (SAM, activator of CBS), but greatly increased by NH2OH. Neither NH2OH, Cys nor SAM had any effect on expression of bcl-2 mRNA in hypoxic medullary slices. These results indicate that endogenously generated H2S was involved in protection of the medullary respiratory centers against hypoxic injury partly via antioxidation and down-regulation of c-fos. [Copyright &y& Elsevier]

Abstract: Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering. [ABSTRACT FROM AUTHOR]

Abstract: During early stage of embryonic development, the liver bud, arising from the foregut endoderm, is the beginning for the formation of future liver three-dimensional structure. While the gene expression profiles associated with this developmental stage have been well explored, the detailed cellular events are not as clear. Epithelial–mesenchymal transition (EMT) was thought to be essential for cell migration in the early vertebrate embryo but seldom demonstrated in human liver development. In this study, we tried to identify the cell populations with both stem cell and EMT features in the human liver bud. Our in situ studies show that the phenotype of EMT occurs at initiation of human liver development, accompanied by up-regulation of EMT associated genes. A human liver bud derived stem cell line (hLBSC) was established, which expressed not only genes specific to both mesenchymal cells and hepatic cells, but also octamer-binding protein 4 (OCT4) and nanog. Placed in appropriate media, hLBSC differentiated into hepatocytes, adipocytes, osteoblast-like cells and neuron-like cells in vitro. When transplanted into severe combined immunodeficiency mice pre-treated by carbon tetrachloride, hLBSC engrafted into the liver parenchyma and proliferated. These data suggests that there are cell populations with stem cell and EMT-like properties in the human liver bud, which may play an important role in the beginning of the spatial structure construction of the liver. [Copyright &y& Elsevier]

Most stem cell therapies involve direct, intraparachymal placement of neural progenitor cells. These cells provide physical support to the endogenous neuronal population and may be engineered to provide in situ growth factor support. Insulin-like growth factor-I (IGF-I) has potent neurotrophic and neuroprotective properties and is expressed by human neural stem cells (hNSCs). IGF-I is implicated in multiple aspects of cell behavior, including proliferation, differentiation, and survival. Enhancing hNSC function through IGF-I overexpression may increase the benefits of stem cell therapy. As a first step to that goal, we examined the direct effects of IGF-I on hNSC behavior in vitro. We demonstrate that IGF-I treatment enhances both the number and length of hNSC neurites. This is correlated with a decrease in proliferation, suggesting that IGF-I promotes neurite outgrowth but not proliferation. While IGF-I activates both AKT and MAPK signaling in hNSCs, we demonstrate that IGF-I-mediated neurite outgrowth is dependent only on AKT signaling. Finally, we demonstrate that IGF-I is neuroprotective after glutamate exposure in a model of excitotoxic cell death. [ABSTRACT FROM AUTHOR]

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/ dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose PBAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae. [ABSTRACT FROM AUTHOR]

Abstract: Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells. [Copyright &y& Elsevier]

Abstract: Hydrogen sulfide (H2S) has been shown to play a protective role in injury of cells induced by hypoxia. Little is known however about its effect on medullary hypoxia-induced rhythmic respiratory suppression. In the present study, a decrease in frequency of rhythmic discharge of hypoglossal rootlets was observed in medullary slices of neonatal rats perfused with 95% N2–5% CO2 to produce hypoxia. Perfusion with NaHS (H2S donor) prevented the inhibitory effect of hypoxia on the burst activity of the rootlets, whereas such action of NaHS was suppressed by pretreatment with glibenclamide, a blocker of KATP channels. In addition, the increase in malondialdehyde content and the up-regulation of c-fos mRNA expression of the slices induced by hypoxia was significantly reduced by NaHS. These results indicate that exogenous H2S may protect the medullary respiratory center against hypoxic injury via activation of KATP channels, reduction of lipid peroxidation and down-regulation of c-fos. [Copyright &y& Elsevier]