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Tamoxifen is a widely used anticancer drug with both an estrogen agonist and antagonist effect. This study focused on its endocrine disrupting effect, and overall environmental significance. Zebrafish embryos were exposed to different concentrations (0.5, 5, 50 and 500 µg l-1) of tamoxifen for 96 h. The results showed a complex effect of tamoxifen on zebrafish embryo development. For the 500 µg l-1 exposure group, the heart rate was decreased by 20% and mild defects in caudal fin and skin were observed. Expressions of a series of genes related to endocrine and morphological changes were subsequently tested through quantitative real-time polymerase chain reaction. Bisphenol A as a known estrogen was also tested as an endocrine-related comparison. Among the expression of endocrine-related genes, esr1, ar, cyp19a1b, hsd3b1 and ugt1a1 were all increased by tamoxifen exposure, similar to bisphenol A. The cyp19a1b is a key gene that controls estrogen synthesis. Exposure to 0.5, 5, 50 and 500 µg l-1 of tamoxifen caused upregulation of cyp19a1b expression to 152%, 568%, 953% and 2024% compared to controls, higher than the effects from the same concentrations of bisphenol A treatment, yet vtg1 was suppressed by 24% from exposure to 500 µg l-1 tamoxifen. The expression of metabolic-related genes such as cyp1a, cyp1c2, cyp3a65, gpx1a, gstp1, gsr and genes related to observed morphological changes such as krt17 were also found to be upregulated by high concentrations of tamoxifen. These findings indicated the potential environmental effect of tamoxifen on teleost early development. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Penile cancer (PeCa) is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis) cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also provides new insights into future investigations aimed to explore the in-depth mechanisms of miRNAs and other small RNAs including piRNAs in penile carcinogenesis regulation and effective target-specific theragnosis. [ABSTRACT FROM AUTHOR]

Major depression is a debilitating disease. To date, the development of biomarkers of major depressive disorder (MDD) remains a challenge. Recently, alterations in the expression of microRNAs (miRNAs) from post-mortem brain tissue and peripheral blood have been linked to MDD. The goals of this study were to detect the differential miRNAs in cerebrospinal fluid (CSF) and serum of MDD patients. First, the relative expression levels of 179 miRNAs (relative high levels in serum) were analyzed by miRNA PCR Panel in the CSF of MDD patients. Then, the differentially altered miRNAs from CSF were further assessed by qRT-PCR in the serum of the same patients. Finally, the serum differentially altered miRNAs were further validated by qRT-PCR in the serum of another MDD patients. The CSF-results indicated that 11 miRNAs in MDD patients were significantly higher than these in control subjects, and 5 miRNAs were significantly lower than these in control subjects. The serum-results from the same patients showed that 3 miRNAs (miR-221-3p, miR-34a-5p, and let-7d-3p) of the 11 miRNAs were significantly higher than these in control subjects, and 1 miRNA (miR-451a) of 5 miRNAs was significantly lower than these in control subjects. The up-regulation of miR-221-3p, miR-34a-5p, let-7d-3p and down-regulation of miR-451a was further validated in another 32 MDD patients. ROC analysis showed that the area under curve of let-7d-3p, miR-34a-5p, miR-221-3p and miR-451a was 0.94, 0.98, 0.97 and 0.94, with specificity of 90.48%, 95.24%, 90.48% and 90.48%, and sensitivity of 93.75%, 96.88%, 90.63% and 84.85%, respectively. In addition, target gene prediction found that the altered miRNAs are involved in affecting some important genes and pathway related to MDD. Our results suggested that differentially altered miRNAs in CSF might be involved in MDD, and serum miR-221-3p, miR-34a-5p, let-7d-3p, and miR-451a might be able to serve as biomarkers for MDD. [ABSTRACT FROM AUTHOR]

Background: PTPRD, encoding protein tyrosine phosphatases receptor type D, is located at chromosome 9p23–24.1, a loci frequently lost in many types of tumors. Recently, PTPRD has been proposed to function as a tumor suppressor gene. The current study aimed to investigate PTPRD expression and its prognostic significance in primary gastric adenocarcinoma. Methods and Results: Quantitative real time reverse transcription PCR (qRT-PCR) and western blotting were used to examine PTPRD expression in paired gastric tumourous and paracancerous tissues. Compared with the matched normal gastric mucosa tissues, both the mRNA (P = 0.0138) and protein (P = 0.0093) expression of PTPRD in fresh surgical specimens were significantly reduced. Clinicopathological and prognostic roles of PTPRD in gastric adenocarcinoma were investigated using immunohistochemistry with 513 paraffin-embedded gastric adenocarcinoma tissue blocks. Statistical analysis revealed that reduced PTPRD expression was significantly associated with T stage (P = 0.004), TNM stage (P<0.001) and tumor size (P = 0.003). Furthermore, Kaplan-Meier survival analysis revealed that low expression of PTPRD significantly correlated with poor survival of gastric cancer patients (P<0.001). Cox regression analysis confirmed PTPRD expression as independent predictor of the overall survival of gastric cancer patients. The MTT assay determined the effects of PTPRD on cell proliferation of MGC803 and GES1 cell lines. Restoring PTPRD expression in MGC803 cells significantly inhibited their growth rate. Silencing PTPRD expression by siRNA treatment in GES1 significantly enhanced cell proliferation compared with mock siRNA treatment. Methylation analysis of PTPRD promoter CpG island in 3 primary GC samples showed one case with partial methylation. Conclusions: These results indicated that PTPRD is a candidate tumour suppressor in gastric cancer. Thus, PTPRD may play an important role in gastric tumorigenesis and serve as a valuable prognostic marker of gastric adenocarcinoma. [ABSTRACT FROM AUTHOR]

The cultivated potato ( Solanum tuberosum L.) is an autotetraploid species. The complexity of tetrasomic inheritance and the lack of pure lines increase the difficulty of genetic analysis of the inherited characteristics. Tuberization is the determinant step for economic yield of potato. To understand the complex genetic basis of tuberization of the cultivated potato, we developed linkage maps for a tetraploid population (F1) of 237 genotypes and mapped QTLs for the percent of in vitro tuberized plantlets (% IVT). The paternal map for E108 (well tuberized) covered 948 cM and included 12 linkage groups, all of which contained all four homologous chromosomes. The maternal map for E20 (nontuberized) covered 1,286 cM and included 14 linkage groups, 12 of which contained all four homologous chromosomes. All 12 chromosomes of potato were tagged using the SSR markers. A major QTL (MT05) with additive effect was detected on chromosome V of E108 which explained 16.23 % of the variation for % IVT, and two minor QTLs (mt05 and mt09) displaying simplex dominant effects were located on chromosome V and chromosome IX of E20 which explained 5.33 and 4.59 % of the variation for % IVT, respectively. Based on the additive model of MT05, the segregation ratio of the gametic genotypes ( Q−: qq = 5:1) matched the ratio of the tuberized genotypes to the nontuberized genotypes in the population suggesting that the segregation of in vitro tuberization in this population is controlled by a major-effect gene or genes. The mapping results of three important candidate genes indicated that the QTL causal genes detected in our study are new. In this study, we developed the almost complete linkage maps of a tetraploid population, identified a major QTL on chromosome V affecting in vitro tuberization, suggested a major-effect gene with minor modifiers model controlling this trait and found that the QTLs identified here correspond to new tuberization genes. Our work provides new and useful information about the genetic basis for tuberization of this autotetraploid crop. [ABSTRACT FROM AUTHOR]

PML/RARA is the oncoprotein driving acute promyelocytic leukemia (APL). It suppresses genes expression by recruitment of a number of transcriptional repressors, resulting in differentiation block and malignant transformation of hematopoietic cells. Here, we found that mice primary hematopoietic progenitor cells (HPCs), transduced by DNA-binding-defective PML/RARA mutants, were deficient in colony formation. Further experiments showed that DNA-binding-defective PML/RARA mutants could not repress the transcription of retinoic acid regulated genes. Intriguingly, there were no significant differences of the micro-speckled intracellular distribution between the mutants and wild-type PML/RARA. Some retinoic acid target genes regulated by PML/RARA are involved in not only differentiation block but also hematopoietic cell self-renewal. Altogether, our data demonstrate that direct DNA-binding is essential for PML/RARA to immortalize hematopoietic cells, while disruption of PML-nuclear body does not seem to be a prerequisite for hematopoietic cell transformation. [ABSTRACT FROM AUTHOR]

Aim:The chemerin receptor CMKLR1 is one type of G protein-coupled receptors abundant in monocyte-derived dendritic cells and macrophages, which plays a key role in the entry of a subset of immunodeficiency viruses including HIV/SIV into lymphocytes and macrophages. The aim of this work was to investigate how CMKLR1 was internalized and whether its internalization affected cell signaling in vitro.Methods:Rat basophilic leukemia RBL-2H3 cells, HEK 293 cells, and HeLa cells were used. CMKLR1 internalization was visualized by confocal microscopy imaging or using a FACScan flow cytometer. Six potential phosphorylation sites (Ser337, Ser343, Thr352, Ser344, Ser347, and Ser350) in CMKLR1 were substituted with alanine using site-directed mutagenesis. Heterologous expression of wild type and mutant CMKLR1 allowed for functional characterization of endocytosis, Ca2+ flux and extracellular signal-regulated kinase (ERK) phosphorylation.Results:Chemerin and the chemerin-derived nonapeptide (C9) induced dose-dependent loss of cell surface CMKLR1-GFP fusion protein and increased its intracellular accumulation in HEK 293 cells and RBL-2H3 cells stably expressing CMKLR1. Up to 90% of CMKLR1 was internalized after treatment with C9 (1 μmol/L). By using different agents, it was demonstrated that clathrin-independent mechanism was involved in CMKLR1 internalization. Mutations in Ser343 for G protein-coupled receptor kinase phosphorylation and in Ser347 for PKC phosphorylation abrogated CMKLR1 internalization. Loss of CMKLR1 internalization partially enhanced the receptor signaling, as shown by increased Ca2+ flux and a shorter latency to peak level of ERK phosphorylation.Conclusion:CMKLR1 internalization occurs in a clathrin-independent manner, which negatively regulated the receptor-mediated Ca2+ flux and ERK phosphorylation. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) are critical regulators of cell differentiation, survival, proliferation, and migration in cancers. This study found that ARNO (cytohesin-2), an activator of the EGF and IGF-I pathways, was more highly expressed in colorectal cancer tissue than in benign adjacent colorectal tissue. When ARNO-siRNA or the chemical inhibitor SecinH3 blocked ARNO, the downstream of the EGF and IGF-I pathways decreased in colorectal cell lines HT29 and HCT116. This blocking also weakened cell proliferation, invasion, and migration in vitro. Furthermore, EGF receptor (EGFR)-dependent colorectal tumor xenografts in nude mouse exerted anti-proliferative and growth suppression effects by injecting secineH3. These data suggested that inhibiting cytohesins or ARNO as cytoplasmic activators of EGFR and IGF-I in colorectal cancer resulted in anti-proliferation, reduced invasion, decreased migration, and suppressed growth in vivo and in vitro. Therefore, cytohesins or ARNO may be a potential therapy target for some colorectal cancer. [ABSTRACT FROM AUTHOR]

The Zinc Finger (ZNF) 280B protein was identified as an unexpected target of an shRNA designed for sGCα1. Further analysis showed that these two proteins are connected in another way, with 280B up-regulation of sGCα1 expression. Knock-down and over-expression experiments showed that 280B serves pro-growth and pro-survival functions in prostate cancer. Surprisingly however, these pro-cancer functions of 280B are not mediated by sGCα1, which itself has similar functions in prostate cancer, but by down-regulated p53. The p53 protein is a second target of 280B in prostate cancer, but unlike sGCα1, p53 is down-regulated by 280B. 280B induces p53 nuclear export, leading to subsequent proteasomal degradation. The protein responsible for p53 regulation by 280B is Mdm2, the E3 ubiquitin ligase that promotes p53 degradation by inducing its nuclear export. We show here that 280B up-regulates expression of Mdm2 in prostate cancer cells, and this regulation is via the Mdm2 promoter. To demonstrate an in vivo relevance to this interaction, expression studies show that 280B protein levels are up-regulated in prostate cancer and these levels correspond to reduced levels of p53. Thus, by enhancing the expression of Mdm2, the uncharacterized 280B protein provides a novel mechanism of p53 suppression in prostate cancer. [ABSTRACT FROM AUTHOR]

Pulsed electromagnetic field (PEMF) has been shown to increase bone mineral density in osteoporosis patients and prevent bone loss in ovariectomized rats. But the mechanisms through which PEMF elicits these favorable biological responses are still not fully understood. Receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) are cytokines predominantly secreted by osteoblasts and play a central role in differentiation and functional activation of osteoclasts. The purpose of this study was to investigate the effects of PEMF on RANKL and OPG expression in ovariectomized rats. Thirty 3-month-old female Sprague-Dawley rats were randomly divided into three groups: sham-operated control (Sham), ovariectomy control (OVX), and ovariectomy with PEMF treatment (PEMF). After 12-week interventions, the results showed that PEMF increased serum 17β-estradiol level, reduced serum tartrate-resistant acid phosphatase level, increased bone mineral density, and inhibited deterioration of bone microarchitecture and strength in OVX rats. Furthermore, PEMF could suppress RANKL expression and improve OPG expression in bone marrow cells of OVX rats. In conclusion, this study suggests that PEMF can prevent ovariectomy-induced bone loss through regulating the expression of RANKL and OPG. [ABSTRACT FROM AUTHOR]

Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The deregulation of E-cadherin is associated with Src/FAK signaling axis and histone deacetylase (HDAC)/EZH2 activity. However, the association between EZH2 and FAK and its clinical significance in endometrial carcinoma has not been reported. Methods: 202 archived cases of endometrial carcinoma (1996–2000) were reviewed and divided into two subtypes. TMAs were developed as per established procedures. EZH2, FAK, and pFAK immunohistochemical stains were performed and the expression was scored as negative (0), low (1) and high (2). Proper statistical analysis was used to assess the correlation between the expression profiles and the clinicopathological parameters and clinical outcome. Results: A total of 141 (69.8%) type-1 tumors and 61 (30.2%) type-2 tumors were identified. EZH2 overexpression was identified in 7.6% of type-1 tumors vs. 63% of type-2 tumors (p<0.001). FAK and pFAK overexpression was only seen in 24.8% and 1.7% of Type-1 tumors as compared to 72% and 58.8% of type-2 tumors, respectively (p<0.001). A positive correlation between the expression of EZH2, FAK, pFAK and PTEN (p<0.0001) was found. The overexpression of EZH2, FAK, and pFAK were significantly associated with high histologic grade, angiolymphatic invasion, lymph node metastasis, myometrial invasion and cervical involvement (p<0.01). Kaplan–Meier analysis demonstrates that the overexpression of EZH2 (p=0.0024), FAK and pFAK (p=0.0001) was significantly associated with decreased overall survival. Conclusion: The overexpression of EZH2, FAK and pFAK correlates with well established pathologic risk factors and may predict a more aggressive biologic behavior in endometrial carcinoma, transforming these proteins into potential therapeutic targets for treatment of endometrial cancer. [Copyright &y& Elsevier]

Background: Compelling evidence suggests that SIRT1, NAD+-dependent class III protein deacetylase, plays an important role in the prevention and treatment of atherosclerosis by counteracting inflammation. Cluster of differentiation 40 (CD40), as a pro-inflammatory cytokine, has been shown to participate in the pathophysiology of atherosclerosis. The relationship between SIRT1 and CD40, however, remained elusive. The present study was thus designed to explore the potential effect of SIRT1 on CD40 expression induced by tumor necrosis factor-α (TNF-α) and to disclose the underlying mechanism in CRL-1730 endothelial cells. Methods: mRNA and protein expressions were identified by quantitative real-time PCR and Western blot respectively. Subcellular localization of SIRT1 was detected by immunofluorescence analysis. SIRT1 small-interfering RNA (siRNA) was carried out for mechanism study. Results: TNF-α reduced SIRT1 expression and induced CD40 expression in CRL-1730 endothelial cells in a time- and concentration- dependent manner. Pretreatment with resveratrol (a potent SIRT1 activator) inhibited TNF-α-induced CD40 expression, while pretreatment with nicotinamide (class b HDACs inhibitor nicotinamide) or sirtinol (a known SIRT1 inhibitor), especially SIRT1 siRNA significantly augmented TNF-α-induced CD40 expression. The frther sudy idicated that PDTC (NF-κB inhibitor) pretreatment attenuated TNF-α-induced CD40 expression, and SIRT1 siRNA significantly augmented TNF-α-induced acetylated-NF-κB p65 (Lys310) expression. Conclusion: The present study provides the direct evidence that SIRT1 can inhibit TNF-α- induced CD40 expression in CRL-1730 endothelial cells by deacetylating the RelA/p65 subunit of NF-κB at lysine 310, which provides new insights into understanding of the anti-inflammatory and anti-athroscerotic actions of SIRT1. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Some of the most serious diseases are characterized by the presence of a specific secondary structure within DNA or RNA, often in the promoter or the coding region of the responsible gene, that enhances or disrupts expression of the protein. Structural elements that impact cellular function may also be formed in other genomic regions such as telomeres. Compounds that interact with such structural elements may be useful in diagnosis or treatment of patients. In this report, we present a FRET melting assay that allows testing of libraries of compounds against four different nucleic acid structures. Compounds are tested to determine whether they stabilize preformed secondary structures (i.e., whether they cause an increase in melting temperature (Tm )). This property is described by the ΔTm parameter, which is the difference between the Tm of the compound-stabilized structure and the Tm of the unbound structure. Model oligonucleotides are labeled with FAM as a fluorescent donor and TAMRA as an acceptor. The intensity of FAM fluorescence is recorded as a function of temperature. Melting temperatures are determined by the FRET method in 96-well plates; this assay could easily be converted into 384-well format. [Copyright &y& Elsevier]

Purpose: Abnormal miRNA expression is associated with prostate cancer progression. However, the relationship between miRNA and biochemical recurrence after radical prostatectomy is not well established. Thus, we evaluated the miRNA miR-21 as a biomarker to predict the risk of biochemical failure, and as a potential drug target for prostate cancer therapy. Materials and Methods: miR-21 levels were assayed using locked nucleic acid in situ hybridization coupled with tissue microarray techniques in 169 radical prostatectomy tissue samples. The Cox proportional hazard model was used to analyze miR-21 expression as an independent predictor of biochemical recurrence. The association of miR-21 with recurrence was estimated using the Kaplan-Meier method. miR-21 was also evaluated as a potential drug target for prostate cancer therapy. Results: miR-21 expression in prostate cancer tissue samples was significantly associated with pathological stage, lymph node metastasis, capsular invasion, organ confined disease, Gleason score, biochemical recurrence and patient followup. Multivariate analysis also indicated that miR-21 expression could be an independent predictor of biochemical recurrence. The 5-year recurrence-free probability for patients positive vs negative for miR-21 expression was 33.9% vs 44.5%. In vivo treatment with antagomir-21 also repressed the tumor growth of DU145 cells in nude mice. Conclusions: Positive miR-21 expression was associated with poor biochemical recurrence-free survival and predicted the risk of biochemical recurrence in patients with prostate cancer after radical prostatectomy. Accordingly gene therapy using miR-21 inhibition strategies may prove useful for prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: Human cytomegalovirus immediate early proteins (CMV IEs) are involved in transcriptional activities of both host and virus gene expression. This study shows that the transcriptional activity of myocardin in regulating cardiomyocyte hypertrophy is enhanced by co-expressing CMV IE2. Forced expression of IE2 increases the augmented cell size of neonatal rat cardiac myocytes induced by myocardin, as well as the mRNA and protein levels of hypertrophic genes, whereas deletion of CArG boxes in the atrial natriuretic factor (ANF) promoter attenuates the effect of CMV IE2 with myocardin. In conclusion, CMV IE2 synergistically stimulates myocardin transactivity in the hypertrophic marker gene ANF in a CArG box-dependent manner. Our study indicates that the association of CMV IEs with myocardin-induced transcription may be involved in myocardin-mediated cardiac hypertrophy. [Copyright &y& Elsevier]

Abstract: Red fluorescent protein (RFP) DsRed and its variants are widely applied in live-cell imaging experiments. However, a major factor that restricts the application of DsRed is its cytotoxicity. Here, we report that DsRed and its variant DsRed-Express2 inhibit the expression of B-cell lymphoma-extra large (Bcl-xL) in HeLa cells by translational regulation. Over-expression of Bcl-xL can reduce the cytotoxicity of DsRed. Meanwhile, Turbo RFP, a mutant RFP from Entacmaea quadricolor, does not affect Bcl-xL expression, suggesting that cytotoxic mechanisms of RFP from different species may be varied. Our results reveal a possible mechanism for the cytotoxicity of DsRed, providing a potential strategy to improve the application of DsRed and its variants. [Copyright &y& Elsevier]

Background: BRAF mutations activate the mitogen-activated protein kinase pathway and often confer an aggressive thyroid cancer (TC) phenotype. Spry2 is an inducible negative feedback regulator of the mitogen-activated protein kinase (MAPK) pathway. The aim of this study was to investigate the role of Spry2 in TC. Methods: TC cell lines were analyzed for Spry2 expression and MAPK pathway activation. Cells were treated with MEK inhibitor and Spry2 small hairpin RNA. Cells were analyzed for Spry2 expression and MEK/ERK phosphorylation (pMEK, pERK). Thirty human papillary TCs were analyzed for mitogen-activated protein kinase pathway activating mutations and Spry2 expression. Results: Increased baseline pMEK levels and Spry2 expression was found in BRAF V600E mutant (BRAF+) cells. MEK inhibition in BRAF+ cells showed decreased Spry2 expression and decreased pMEK/pERK levels. From our tissue samples, 10 papillary TCs had BRAF mutation, and increased Spry2 expression was found only in BRAF+ tumors. Conclusion: Spry2 expression correlates with BRAF status in vitro and in human tissue. Spry2 may serve as a negative feedback regulator of the mitogen-activated protein kinase pathway in BRAF+ TC. Increased Spry2 expression may serve as a surrogate marker of mitogen-activated protein kinase pathway activation with prognostic and therapeutic implications. [ABSTRACT FROM AUTHOR]

Abstract: Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: The white shrimp Litopenaeus vannamei is one of the most important economic species in shrimp farming and are frequently exposed to multiple stressors (including temperature) in aquaculture. The differential expression of seven genes encoding antioxidant enzymes and stressor biomarkers was investigated by real-time quantitative PCR in haemocytes and hepatopancreas and gill extracted from L. vannamei following acute temperature stress. Temperature stress induced CAT, GST, ferritin and HSP60 gene expression in gills. Western blot results also revealed that HSP60 was induced in the same tissue after acute temperature stress. Heat stress resulted in an increase in most examined genes in haemocytes (excluding HSP60) suggesting haemocytes may be an early response tissue in acute temperature stress. GST was significantly up-regulated in haemocytes (to up to 16.4 fold at 22°C and 71.8 fold at 28°C, respectively) during exposure to heat stress. In addition, MnSOD was more strongly induced in haemocytes and hepatopancreas (to up to 273.8 fold and 115.8 fold, respectively) after exposure to 28°C from 15°C implying their important role in antioxidant protection in response to heat stress. The transcriptional responses of these genes to temperature stress will provide the basis for a multi-biomarker system that could be used for the biomonitoring of aquatic environments. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, cDNA encoding a heat shock protein 60 (LvHSP60) gene in Litopenaeus vannamei was cloned using a combination of homology and rapid amplification of cDNA end (RACE) methods. The full length of the LvHSP60 cDNA was found to be 2379bp, with a 1737bp open reading frame. The translated amino acid sequence consisted of 579 residues with a calculated molecular mass of 60.8kD and an isoelectronic point (pI) of 5.97. Comparison of the deduced amino acid sequence showed that it has high identity (85–89%) with HSP60/chaperonins from insects and mammals. Quantitative real-time PCR and Western blot analysis were carried out to investigate the expression patterns and distribution profiles of LvHSP60 before and after stimulation with the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Vibrio alginolyticus. LvHSP60 mRNA was found to be both constitutive and inducible, and was highly expressed in haemocytes and almost all tissues examined, including muscle, stomach, heart, hepatopancreas and gill tissue, but it was less strongly expressed in the intestine. The expression analysis revealed that LvHSP60 was significantly up-regulated in the gills, hepatopancreas and haemocytes after bacterial challenge. Transcription of LvHSP70 was also induced in haemocytes and the hepatopancreas after different bacteria injection. Subsequent flow cytometry analysis showed that the concentration of Ca2+ ions increased significantly within bacteria-challenged haemocytes by 1.5h after injection. The results indicate that LvHSP60 and LvHSP70 may play important roles in mediating the immune responses of L. vannamei to bacterial challenge, and that the Ca2+ signalling transduction pathway may be involved in the initiation of the shrimp’s immune responses in early stages of infection. [Copyright &y& Elsevier]

Abstract: The ROS production, the percentage of dead and damaged haemocytes, the DNA Olive Tail Moment (OTM) value and the gene expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx), were studied in the Pacific white shrimp, Litopenaeus vannamei, when exposed to acute pH stress. The increased ROS production in haemocytes and the increased OTM value in both the haemocytes and the hepatopancreas cells suggest that oxidative damage occurred in shrimp exposed to pH 5.6 and pH 9.3, with apoptosis, mainly being associated with excess Ca2+influx and changes in cell viability. Acid and alkaline pH-induced DNA damage was time dependent in the haemocytes and the hepatopancreas cells. The concentration of intracellular free calcium [Ca2+] i after different pH treatments increased significantly over time, reaching its highest concentration after 12 h, but decreasing gradually to normal levels after 24 h. The [Ca2+] i content in shrimp cells when exposed to pH 9.3 and pH 5.6 for 12 h had increased by 58%–81%, compared with exposure to pH 7.4 (control). In addition, the gene expression of cMnSOD, CAT, GPx and TRx in the hepatopancreas of L. vannamei was induced by acid and alkaline pH stress, although there were differences in the expression response with respect to the duration of induction and the different pH treatments (acid or alkaline). Our results show that acidic or alkaline-induced oxidative stress may cause DNA damage, and cooperatively activate expression of CAT, GPx and TRx mRNA. Calcium ions appear to be important in mediating shrimp responses to pH stress. [Copyright &y& Elsevier]

Abstract: To investigate the effects of selenium on mRNA expressions of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) in the pancreas of streptozotocin-induced diabetic mice, the animals were divided into three groups in this study: a normal control group, an untreated diabetes mellitus group and a selenite-treated diabetes mellitus group. Selenite was administered to the diabetic mice in selenite-treated diabetes mellitus group for 2 weeks with an oral dose of 2 mg/kg body weight per day by gavage. The results showed that pancreatic selenium content and glutathione peroxidase mRNA expression and activity were decreased by 16.0%, 63.9% (P<.01) and 31.2 % (P<.01), respectively, in untreated diabetes mellitus group compared with normal control group, and they were significantly increased by 51.0% (P<.001), 79.7% (P<.05) and 21.0% (P<.05), respectively, in selenite-treated diabetes mellitus group compared with untreated diabetes mellitus group. Meanwhile, pancreatic mRNA expressions of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α and interferon-gamma; mRNA expression and activity of iNOS and content of nitric oxide were significantly increased by 133.0% (P<.01), 164.0% (P<.001), 111.0% (P<.01), 101.0% (P<.001), 73.2% (P<.001) and 37.6% (P<.01), respectively, in untreated diabetes mellitus group compared with normal control group, and they were decreased by 43.2% (P<.01), 37.5% (P<.01), 33.9 % (P<.05), 35.5% (P<.01), 34.9% (P<.01) and 18.1% (P<.05), respectively, in selenite-treated diabetes mellitus group compared with untreated diabetes mellitus group. In conclusion, the chosen pharmacological dose of selenium provides partial correction of these effects towards control values. Moreover, the results suggested that the hypoglycemic role of selenium may relate with its inhibiting effect on augmentation of proinflammatory cytokines and reactive oxygen species/reactive nitrogen species by streptozotocin inducing in the pancreas of diabetic mice. [Copyright &y& Elsevier]

Abstract: Ferritin is a storage protein that plays a key role in iron metabolism. In this study, the gene expression of ferritin in hepatopancreas, muscle and eye of white shrimp Litopenaeus vannamei were analysed by quantitative real-time PCR after exposure to pH stress. The results showed the expression level in the hepatopancreas and eye were about 5 times to those of beta-actin, whereas lower expression levels in muscle were about 1/2 to those of beta actin. The gene expressions of ferritin in hepatopancreas and muscle were up regulated at 1.5 h after exposure to pH stress, but down-regulated in eye slightly. The ferritin transcript in hepatopancreas of white shrimp L. vannamei at pH 9.3 and pH 5.6 treatments increased significantly in 1.5 h and 12 h post-challenge respectively, but both returned to the original values in 24h post-challenge. The present result showed that ferritin expression in tissue of L.vannamei was induced by acute pH stress and ferritin induction varied in response to duration of induction and acidic or alkaline pH treatment, and was more sensitive to high pH than low pH. [Copyright &y& Elsevier]

Background. ANLN and miR-30a-5p may be involved in the progression of lung adenocarcinoma (LUAD). However, their underlying mechanism in LUAD has not been completely comprehended. Methods. Differential expression analysis, binding site prediction, and survival analysis were conducted by bioinformatics approaches. ANLN mRNA and miR-30a-5p expression were detected by qRT-PCR. ANLN protein expression was detected by western blot. Cell behaviors in LUAD were examined by functional experiments. Results. ANLN was activated in LUAD cells in terms of mRNA and protein. High ANLN level was positively correlated with poor prognosis. Enforced ANLN stimulated protumorigenesis LUAD cell behaviors. miR-30a-5p could target ANLN mRNA, as revealed and verified through assays. Remarkably low miR-30a-5p expression was observed in LUAD cells, and it could repress ANLN expression. The accelerated cell behaviors by overexpression of ANLN were counteracted by upregulating miR-30a-5p. Conclusion. Overall, miR-30a-5p remarkably restrained the malignant progression of LUAD cells by constraining ANLN expression. Thus, ANLN and miR-30a-5p could be novel therapeutic targets of LUAD. [ABSTRACT FROM AUTHOR]

Background: Wenshen Jianpi recipe (WSJPR), a blended traditional Chinese medicine, is considered to have the possible beneficial effect on the progression of diabetic nephropathy (DN). This present study was designed to elucidate this protective activity in a rat model with streptozotocin (STZ)-induced DN and to explore the possible underlying mechanism. Methods: Adult Sprague Dawley (SD) rats were induced to develop DN through intraperitoneal injection of STZ (60 mg/kg). Animals were orally administered saline, WSJPR at 7.5, 15, 30 g/kg, and valsartan (25 mg/kg) daily for 8 weeks. Blood and 24-h urine samples of each rat were collected for biochemical examination at 2-week intervals. Microcirculatory blood flow in the renal cortex and hemorheology index were also measured. At the end of 8 weeks, all rats were sacrificed to obtain the kidney tissues for histological examination and reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the transcriptional levels of nephrin and podocin genes. Results: WSJPR could improve serum total protein (TP) and albumin (ALB), reduce the excretion rates of urine-TP (U-TP), urine-ALB (U-ALB) and urine urea nitrogen (UUN) (P < 0.05), although it did not significantly alter the hyperglycemia. In addition, treatment with WSJPR could strongly reduce blood flow, erythrocyte aggregation index, and ameliorate microcirculation. In histological measurement, WSJPR-treated rats showed a significant amelioration in glomerular hypertrophy and mesangial expansion. By RT-PCR, we found WSJPR up-regulated the nephrin and podocin expression at mRNA levels. Conclusion: This study suggested that WSJPR could effectively relieve renal damage and improve renal function of DN rats by ameliorating metabolism disorder and increasing the gene expression of nephrin and podocin, which might be a useful approach for the treatment of DN. [ABSTRACT FROM AUTHOR]

Background: The yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies. Results: In this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the β-1,4-endoxylanase XynCDBFV or the conventional EGFP. Conclusions: Our results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target. [ABSTRACT FROM AUTHOR]

RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes. [ABSTRACT FROM AUTHOR]

Grass carp (Ctenopharyngodon idella) is the largest economic fish in freshwater culture in China, which is predisposed to infectious diseases under high temperature. Under the background of global warming, the industrialization of the Pearl River Delta region has led to aggravated thermal pollution, which has increasingly serious impacts on the aquatic ecological environment. This will result in more frequent exposure of grass carp to overheated water temperatures. Previous studies have only identified the regulatory genes of fish that respond to pathogens or temperature stress, but the transcriptional response to both is unknown. In this study, the histopathological analysis showed heat stress exacerbated spleen damage induced by Aeromonas hydrophila. The transcriptional responses of the spleens from A. hydrophila lipopolysaccharide (LPS) - injected grass carp undergoing heat stress and at normal temperatures for 6, 24, and 72 h were investigated by mRNA and microRNA sequencing. We identified 28, 20, and 141 differentially expressed (DE) miRNAs and 126, 383, and 4841 DE mRNAs between the two groups after 6, 24, and 72 h, respectively. There were 67 DE genes mainly involved in the cytochrome P450 pathway, antioxidant defense, inflammatory response, pathogen recognition pathway, antigen processing and presentation, and the ubiquitin-proteasome system. There were 5 DE miRNAs involved in regulating apoptosis and inflammation. We further verified 17 DE mRNAs and 5 DE miRNAs using quantitative real-time PCR. Based on miRNAs and mRNAs analysis, continuous heat stress will affect the antibacterial responses of grass carp spleens, resulting in aggravation of spleen injury. Together, these results provide data for further understanding of the decreased tolerance of fish to pathogen infection in persistent high-temperature environments. • Continuous heat stress exacerbates spleen damage induced by Aeromonas hydrophila. • Continuous heat stress impaired the antioxidant and immune functions of grass carp. • HSPs are induced to persist after co-treatment with LPS and heat stress. • MicroRNAs mediate apoptosis and inflammation in the damaged spleen of grass carp. [ABSTRACT FROM AUTHOR]

The study was a microarray (Applied Biosystems, AB) on cecal cells from rats to see effects of resistant starch (RS) on expression of the whole genome: control (CC, 3.6 kcal/g), energy control (EC, 3.2 kcal/g), and RS (3.2 kcal/g, high amylose cornstarch). Male Sprague Dawley (8 wk) rats (RS, n=10; CC and EC, n=9) fed 4 wk. A gene's expression was "present" if signal/noise ≥3. Gene lists were done in R Scripts (t-tests), CC-EC, CC-RS, EC-RS. Lists were >10,000 genes. Differences (p<0.05; <0.01) were: CC-EC, 630; 158; CC-RS, 3193; 1769; EC-RS, 2641; 1301 genes. The Protein Analysis through Evolutionary Relationships of AB was used to determine the number of pathways (CC-EC, 18; CC-RS, 23; EC-RS, 17) affected greater than by random chance (p<0.05); and genes not known in a pathway (CC-EC, 515; CC-RS, 2711; EC-RS, 2266). A large number of hormone genes were affected (CC-EC, 18; CC-RS, 34; EC-RS, 26). Spotfire® was used for clustering analyses. Genes that were up-regulated (down-regulated) by RS were down-regulated (up-regulated) by CC and EC. Cells obtained from the cecum would be a heterogeneous mix therefore, gene expression for RS should result in signaling from the gut to the rest of the body via endocrine, neural, and immune mechanisms. These effects by RS may lead to a healthier phenotype, and decreased risk for chronic diseases such as heart disease, diabetes, and cancer. [ABSTRACT FROM AUTHOR]

This study examined the effects of resistant starch in a high fat diet (28% fat) on body weight, abdominal fat, cecal weight and pH, serum gut peptide YY (PYY) and gene expression for PYY and proglucagon in male Sprague Dawley rats. Three groups of rats (8wk) were fed one of these diets for ten wks: energy control (EC; 3.7 kcal/g), resistant starch (RS; 3.7 kcal/g) and fiber control (FC; 2.9 kcal/g). Results were significant when p≤ 0.05. The consumption of RS resulted in significant increases in cecal weight (full and empty), serum PYY, gene expression for PYY and proglucagon in cecum, and significant decrease in cecal pH compared to the other groups. However the disemboweled body weight and abdominal fat for RS were not lower compared to the EC. In previous studies, we observed that RS in a low fat diet (18% of fat), produced similar gut signaling, as in current study, but reduced disemboweled body weight and abdominal fat compared to EC. Mechanism for the contradictory results needs further study. Since high fat diets have been shown to alter brain response to other satiety signals (leptin), a high fat diet might affect neuronal response to PYY and GLP-1 signaling from the gut caused by dietary RS. [ABSTRACT FROM AUTHOR]

Many regulators controlling arterial identity are well described; however, transcription factors that promote vein identity and vascular patterning have remained largely unknown. We previously identified the transcription factors Islet2 (Isl2) and Nr2f1b required for specification of the vein and tip cell identity mediated by notch pathway in zebrafish. However, the interaction between Isl2 and Nr2f1b is not known. In this study, we report that Nr2f2 plays minor roles on vein and intersegmental vessels (ISV) growth and dissect the genetic interactions among the three transcription factors Isl2, Nr2f1b, and Nr2f2 using a combinatorial knockdown strategy. The double knockdown of isl2/nr2f1b , isl2/nr2f2 , and nr2f1b/nr2f2 showed the enhanced defects in vasculature including less completed ISV, reduced veins, and ISV cells. We further tested the genetic relationship among these three transcription factors. We found isl2 can regulate the expression of nr2f1b and nr2f2 , suggesting a model where Isl2 functions upstream of Nr2f1b and Nr2f2. We hypothsized that Isl2 and Nr2f1b can function together through cis -regulatory binding motifs. In-vitro luciferase assay results, we showed that Isl2 and Nr2f1b can cooperatively enhance gene expression. Moreover, co-immunoprecipitation results indicated that Isl2 and Nr2f1b interact physically. Together, we showed that the interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish. • The interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish. • Physical interaction between Isl2 and Nr2f1b by using genetic and biochemical approaches. • Isl2 and Nr2f1b can cooperatively enhance gene expression via cis -regulatory element of target genes. [ABSTRACT FROM AUTHOR]

The intestine is a main organ responsible for energy and nutrition absorption and digestion and is an important immune barrier in fish. In recent years, the intestinal health of fish has gained increasing attention in the aquaculture industry. The intestine is a direct target organ of starvation and is highly sensitive to nutritional changes. However, the adaptive responses of the fish intestine to starvation are not well studied. The present study aimed to reveal the effects of starvation on antioxidant systems, autophagy, and mitochondrial function in the intestine of Chinese perch. Therefore, Chinese perch (250 ± 22 g) were starved for 0, 7, and 14 days. Our results indicate that the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly increased in the intestine of Chinese perch after 7 days and 14 days of starvation, indicating that starvation could induce oxidative stress in the intestine. Meanwhile, the activities and mRNA expression of antioxidative enzymes, including superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalases (CAT), were upregulated in the intestine after the starvation treatment, which might be a compensatory response to oxidative stress. Starvation also increased the expression of nrf2 in the intestine, thereby resulting in the upregulation of antioxidative gene expression. However, the glutathione content and its generation in the intestine were decreased under starvation stress. The ultrastructural analysis of the intestine demonstrated that starvation induced autophagy in the intestine of Chinese perch. The RT–qPCR analysis showed that starvation induced the mRNA expression of genes involved in autophagosome membrane initiation, autophagosome membrane expansion, vesicle recycling, and cargo recruitment, including beclin1, ulk1, beclin1, lc3a, atg4, atg5, atg7, p62, and atg9. Mitochondria are the main ATP-generating organelles and target organelles for ROS. The present study indicates that starvation induced a decrease in the ATP content in the intestine of Chinese perch. In addition, starvation downregulated the expression of mitochondrial oxidative phosphorylation-related and mitochondrial ATP generation-related genes (cox1, atp5f1, atp5a1, and mt-cyb) in the intestine of Chinese perch, suggesting that starvation could suppress mitochondrial energy synthesis. Taken together, our data demonstrate that starvation can induce oxidative stress, disrupt antioxidant systems, and cause autophagy and mitochondrial dysfunction in the intestine of Chinese perch. These results provide insight into the mechanisms by which starvation disrupts intestinal homeostasis in fish. • Starvation induce oxidative stress, excess autophagy and mitochondrial dysfunction in the intestine of Chinese perch. • Starvation up-regulating activities and mRNA expression of antioxidative enzymes and impaired GSH regeneration in the intestine. • Intestine ATP content and mRNA expression of mitochondrial energy metabolism were decreased by starvation. • Starvation induced the expression of autophagy related genes involving in the autophagosome membrane initiation, autophagosome membrane expansion vesicle recycling and cargo recruitment of autophagy process. [ABSTRACT FROM AUTHOR]

Abstract: Potato microtuber produced in vitro provides a model system to investigate photoperiod-dependent tuberization. However, the genes associated with potato tuberization remain to be elucidated. The present research involved three potato clones with distinct tuberization response to changes of photoperiod. Digital Gene Expression (DGE) Tag Profiling analysis of the short-day-sensitive clone identified 2218 genes that were regulated by day length. Both GO and KEGG pathway analysis provided insights into predominant biological processes and pathways, and enabled the selection of 56 genes associated with circadian rhythmicity, signal transduction, and development. Quantitative transcriptional analysis in the selected clones revealed 5 genes potentially associated with photoperiodic tuberization, which were predicted to encode a DOF protein, a blue light receptor, a lectin, a syntaxin-like protein, and a protein with unknown function. Our results strongly suggest that potato tuberization may be largely controlled by the homologs of genes shown to regulate flowering time in other plants. [Copyright &y& Elsevier]

Hormone receptor-positive breast cancer (HR+ BC) is known to be relatively insensitive to chemotherapy, and since chemotherapy has remained the major neoadjuvant therapy for HR+ BC, the undetermined mechanism of chemoresistance and how chemotherapy reshapes the immune microenvironment need to be explored by high-throughput technology. By using single-cell RNA sequencing and multiplexed immunofluorescence staining analysis of HR+ BC samples (paired pre- and post-neoadjuvant chemotherapy (NAC)), the levels of previously unrecognized immune cell subsets, including CD8+ T cells with pronounced expression of T-cell development (LMNA) and cytotoxicity (FGFBP2) markers, CD4+ T cells characterized by proliferation marker (ATP1B3) expression and macrophages characterized by CD52 expression, were found to be increased post-NAC, which were predictive of chemosensitivity and their antitumor function was also validated with in vitro experiments. In terms of immune checkpoint expression of CD8+ T cells, we found their changes were inconsistent post-NAC, that LAG3, VSIR were decreased, and PDCD1, HAVCR2, CTLA4, KLRC1 and BTLA were increased. In addition, we have identified novel genomic and transcriptional patterns of chemoresistant cancer cells, both innate and acquired, and have confirmed their prognostic value with TCGA cohorts. By shedding light on the ecosystem of HR+ BC reshaped by chemotherapy, our results uncover valuable candidates for predicting chemosensitivity and overcoming chemoresistance in HR+ BC. • Landscape of HR+ BC during chemotherapy at the single-cell level was uncovered. • Novel subsets of immune cells predictive of chemosensitivity were identified. • CD8+ T cells and CD52+ macrophages are more proximate to cancer cells in chemo-sensitive patients. • The expression of checkpoint markers on CD8+ T cells were inconsistently changed post-NAC. • Innate and acquired signatures of chemoresistance have prognostic value for BC patients. [ABSTRACT FROM AUTHOR]

Celastrus orbiculatus Thunb. is an ancient traditional Chinese herb with a long history of medicinal use. The ethyl acetate extract of Celastrus orbiculatus Thunb. (COE) has been shown to have anti-tumor effects in various preclinical studies. However, the anti-invasive and metastatic efficacy of COE in non-small cell lung cancer (NSCLC) and the mechanism by which COE regulates cellular oxidation levels are yet to be elucidated. To study the anti-dissemination effect of COE on NSCLC and to elucidate the molecular mechanism of COE in regulating cellular oxidation levels and its effect on lung cancer invasion and metastasis. CCK-8 assay was used to detect the toxic effects of COE on NSCLC. Transwell assay and high-content imaging was used to detect the Motility of NSCLC. Transmission electron microscopy and three-dimensional (3D) imaging of mitochondrial fluorescence were employed to detect the number and structure of mitochondria. JC-1 probe was used to detect the level of mitochondrial membrane potential. Firefly luciferase assay was used to detect the level of total intracellular ATP. MitoSox probe and DCFH-DA probe were applied to detect the level of reactive oxygen species (ROS) inside the mitochondria and the total intracellular ROS, respectively. Immunohistochemistry was used to detect protein expression in xenograft tumors. COE inhibited motility and induced DJ-1 downregulation in NSCLC at low toxic concentrations, and the antiseptic effect of COE was reduced significantly after the overexpression of DJ-1. COE induced structural disruption of mitochondria in NSCLC and accumulation of superoxide compounds, decreased the volume of membrane potential depolarization, and impaired energy production, ultimately leading to a large accumulation of ROS at the cellular level. The antioxidant acetylcysteine (NAC) significantly reversed the antiseptic capacity of COE. In a xenograft tumor model, protein expression of DJ-1, E-cadherin, N-cadherin, and MMP-2 in COE group was significantly changed compared to the model group. In the present study, COE inhibited NSCLC invasion and metastasis and was associated with the downregulation of DJ-1 and elevated ROS. COE-mediated downregulation of DJ-1 may be the primary cause of mitochondrial structural and functional dysfunction in NSCLC, eventually leading to ROS accumulation. [Display omitted] [ABSTRACT FROM AUTHOR]

Peripheral nerve injury is encountered quite commonly in the clinic, and treatment results are often not satisfactory. Therefore, promoting nerve regeneration and functional recovery is a primary goal of neuroscience research. Recovery of corresponding target muscle can differ following peripheral nerve injury, but the reasons are unknown. Herein, we investigated differential gene and protein expression in gastrocnemius and tibialis anterior muscle following tibial and common peroneal nerve injury using RNA sequencing and proteomics approaches, and analysed the results by bioinformatics. In total, 1794, 1765, 1656 and 2006 differential genes and 398, 400, 959 and 472 differential proteins were identified in gastrocnemius and tibialis anterior muscles at 1, 7, 14 and 21 days after surgery, related to activation of 51 signalling pathways. Differential expression of these genes and proteins may contribute to the degree of recovery of target organs following peripheral nerve injury. The findings provide a foundation for investigating regeneration mechanisms following peripheral nerve injury. • It is first to explore the expression of genes and proteins in the corresponding target muscles after nerves injury. • It is first to explore the differential expression of genes and proteins in tibial and common peroneal nerves after injury. • It explores the expression of differential genes and proteins by RNA sequencing and proteomics techniques. • It suggests that different nerves have different gene and protein expressions in respective muscle tissues after injury. [ABSTRACT FROM AUTHOR]

The dorsal root ganglion (DRG) is actively involved in the development of neuropathic pain (NP), serving as an intermediate station for pain signals from the peripheral nervous system to the central nervous system. The mechanism by which DRG is involved in NP regulation is not fully understood. The immune system plays a pivotal role in the physiological and pathological states of the human body. In recent years, the immune system has been thought to play an increasingly important role in the pathogenesis of NP. The immune system plays a key role in pain through specific immune cells and their immune-related genes (IRGs). However, the mechanism by which IRGs of DRG regulate NP action has not been fully elucidated. Here, we performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of IRGs in DRG bulk-RNA sequencing data from spared nerve injury (SNI) model mice and found that their IRGs were enriched in many pathways, especially in the immune response pathway. Subsequently, we analyzed single-cell RNA sequencing (scRNA-seq) data from DRGs extracted from the SNI model and identified eight cell populations. Among them, the highest IRG activity was presented in macrophages. Next, we analyzed the scRNA and bulk-sequencing data and deduced five common transcription factors (TFs) from differentially expressed genes (DEGs). The protein-protein interaction (PPI) network suggested that Atf3 and JunB are closely related. In vitro experiments, we verified that the protein and mRNA expressions of Atf3 and JunB were up-regulated in macrophages after lipopolysaccharide (LPS) stimulation. Moreover, the down-regulation of Atf3 reduced the release of inflammatory cytokines and decreased the protein and mRNA expression levels of JunB. The down-regulation of JunB also reduced the release of inflammatory cytokines. Furthermore, overexpression of JunB attenuated the effect of Atf3 down-regulation in reducing the release of inflammatory cytokines. Therefore, we speculated that Atf3 might promote NP through JunB-mediated release of inflammatory factors in DRG macrophages. [ABSTRACT FROM AUTHOR]

Abstract G-quadruplexes (G4s) are fascinating non-canonical nucleic acid structures that attract broad attention due to their vital roles in gene expression and various applications. G4s exhibit high structural polymorphism and high resolution techniques (NMR, Crystallography) are often required to unambiguously determine G4 topology. Fast and handy techniques such as UV and CD provide interesting structural information. Here, we monitored the thermal denaturation profiles (TDPs) of various G4s at different wavelengths by UV, and confirmed that all G4 topologies (parallel, antiparallel and hybrid) exhibited hyperchromism at 243 nm and hypochromism at 295 nm. Furthermore, the absorption at 260 nm and 275 nm showed a remarkable topology-dependent behavior, and the hypochromism at 275 nm allows to discriminate parallel conformations from other G4 topologies. Therefore, this work provides a new inexpensive identification method for parallel G4 structures as well as a new perspective for the relationship between TDPs and G4 structures. Graphical abstract Image 1 Highlights • We firstly discovered that the thermal denaturation profiles of parallel G-quadruplexes showed hypochromism at 275 nm. • The thermal denaturation profiles of G-quadruplexes at 260 and 275 nm are strongly structure-dependent. • UV thermal denaturation profiles can confirm the formation of parallel G-quadruplexes directly. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Cerebrovascular diseases (CBVDs), characterized by striking morbidity and mortality, have become the most common life-threatening diseases. The existing drugs of CBVDs target one or a few of pathogenic factors, the efficacy of which is limited because of the complexity of CBVDs. Traditional Chinese medicine (TCM), featured by multi-component and multi-target endows the great effectiveness in CBVDs treatment. For instance, Erigeron breviscapus (vant.) Hand. Mazz. （Erigeron breviscapus) has been used to treat CBVDs for a long time and the efficacy has been verified through years’ of practice. Nevertheless, the mechanisms of Erigeron breviscapus for treating CBVDs are still unclear. The aim of the study Systematically decipher the mechanisms of Erigeron breviscapus for treating CBVDs. Materials and methods The systems pharmacology approach is utilized by integrating ADME pharmacokinetic screening, target fishing, protein-protein interaction (PPI), network analysis and in vitro experiments verification. Results First, 14 potentially active molecules were screened out through in silico ADME pharmacokinetic evaluation, most of which have been reported with excellent biological activities. Then 169 targets of active molecules were read out using our in-house softwares, systems drug targeting (sysDT) and Weighted Ensemble Similarity(WES). We found that the targets of the active compounds were significantly enriched to the CBVDs therapeutic targets by analyzing their biological processes and protein-protein interactions (PPIs). A multi-layer network analysis including compound-target network, target-pathway network and “CBVDs pathway” indicated that the Erigeron breviscapus exerts a protective effect on CBVDs via regulating multiple pathways and hitting on multiple targets. Meanwhile in vitro experiments confirmed that the stigmasterol, scutellarein, and daucosterol from Erigeron breviscapus increased the MEK and PLCγ proteins levels, and decreased the expression of Bax, PI3K, and eNOS, which led to the cell survival, proliferation and contraction. Conclusion The approach used in this work offers a new exemplification for systematically understanding the mechanisms of herbal medicines, which will give an impulse to the CBVDs drug development. [ABSTRACT FROM AUTHOR]

Type II crustins are the most abundant type of crustins in shrimps that exhibit remarkable sequence diversities and broad antibacterial activities. This study characterized a novel type II crustin, SpCrus2 , in the mud crab Scylla paramamosain . The SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a 164-amino acid protein. In the deduced protein, a 17-amino acid signal peptide, a glycine-rich hydrophobic region (GRR), and a cysteine-rich region (CRR) containing a whey acidic protein domain were predicted. Sp Crus2 shares high similarity with most type II crustins (types IIa and IIb crustins) in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins. Sp Crus2 and Pl Crus3 from Pacifastacus leniusculus share high similarity and the same distribution pattern of cysteine residues. Thus, we proposed them as type IIc crustins. SpCrus2 is mainly distributed in the gills and can be up-regulated through Vibrio parahemolyticus or Staphylococcus aureus challenge. To investigate the biological functions of Sp Crus2 and the underlying mechanisms, Sp Crus2, GRR, CRR, and the mutant of CRR (CRR-M, the cysteine distribution pattern is mutated into that in most conventional crustins) were all overexpressed and purified. Sp Crus2 GRR itself, as a glycine-rich amphiphilic peptide, exhibited evident antibacterial ability against Gram-negative bacteria, whereas CRR possessed potent antibacterial activity against Gram-positive bacteria. Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of Sp Crus2, indicating that GRR and CRR synergized to exert their potential antibacterial functions. In addition, CRR exhibited slightly stronger antimicrobial activity than CRR-M, suggesting that Sp Crus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues. The likely antimicrobial ability of Sp Crus2 may result from its microbial polysaccharide-binding and agglutination activities. Overall, this study characterized the first type II crustin in crabs and provided new insights into understanding the sequence and functional diversity of crustins and their immune functions in crustaceans. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance For many years, Guangzhou University of Chinese Medicine has been successfully using the empirical Wenyang Huoxue Jiedu formula (WHJF) to treat coronary heart disease. Modern theories of acute coronary syndrome mainly focus on rupture of thin-cap fibroatheromas (TCFAs), which is closely related to the release of vascular endothelial growth factor and its receptor (VEGF/VEGFR). Aim of study We investigated the effects of WHJF on the formation of TCFA plaques and the potential mechanism (VEGF/VEGFR signaling pathway). Materials and methods For the in vivo experiments, WHJF was administered to ApoE -/- mice, as a model of TCFA plaque formation. Aortic sections of the mice were obtained, and the vulnerability index and new vessel density of plaques were calculated by the Movat staining assay and immunohistochemistry kit, respectively. Protein and mRNA expression levels of VEGF/VEGFR in aortas were assayed by capillary electrophoresis immunoassay and quantitative real-time polymerase chain reaction analyses. In vitro , WHJF serum was produced in rats on the fourth day 2 h after the first administration of different concentrations of WHJF. Proliferation, migration, and lumen formation ability of human umbilical vein endothelial cells (HUVECs) treated with sera from these rats were assayed by the CKK-8 kit, Transwell plates, and Matrigel assay, respectively. Protein and mRNA expression levels of signaling molecules in the VEGF/VEGFR pathways were also examined. Results In vivo , the vulnerability index and new vessel density of plaques in the WHJF group were lower than those values in the blank control group ( P  < 0.05). No differences were found between the groups in the expression levels of VEGF/VEGFR ( P  > 0.05). In vitro , the proliferation, migration, and tube formation of HUVECs in the high-dose WHJF group were reduced compared to the control group ( P  < 0.05). This finding was in agreement with the downregulation of VEGFR-2 and pERK ( P  < 0.05). The mRNA expression of signaling molecules showed no difference between the groups ( P  > 0.05). Conclusions WHJF inhibits TCFA formation by influencing the VEGF/VEGFR signaling pathway. [ABSTRACT FROM AUTHOR]

Transcriptional cofactor Vestigial-like 4 (VGLL4) was considered to take part in the early stage of development. Different from human, three paralogs of vgll4 were found in zebrafish, which were vgll4a , vgll4b and vgll4l . However, the expression patterns of the three paralogs during zebrafish development remains unknown. In this study, we used in situ hybridization to elucidate the temporal and spatial expression of zebrafish vgll4 paralogs during normal embryonic and larval development. Similar expression was shown in certain areas at similar stages for the three paralogs. Expression of vgll4a , vgll4b and vgll4l were all found in pectoral fins and otic vesicles during the early developmental stages. On the other hand, a few differences of the three paralogs expression were found in eyes, pharynx, pharyngeal arches and brain tissues. The expression of vgll4a was weak and ubiquitous, while vgll4b was obviously expressed in brain tissues and vgll4l was clearly restricted to each pair of pharyngeal pouches. What's more, vgll4b and vgll4l had unique expression at mature lateral line neuromasts and forerunner cells respectively. Despite the conservativeness of functional domains, the three paralogs of zebrafish vgll4 shared several similarities and displayed some distinctions in the expression patterns, indicating that they may still have different and exclusive functions, which need to be further explored. [ABSTRACT FROM AUTHOR]

Tau pathology is a pathological hallmark for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia. As a novel susceptibility gene for these 2 diseases, triggering receptor expressed on myeloid cells 2 ( TREM2 ) gene encodes an immune receptor that is uniquely expressed by microglia. Recently, a correlation between TREM2 expression and hyperphosphorylated tau has been revealed in the brain of Alzheimer's disease patients, suggesting a potential association between TREM2 and tau pathology. However, the role of TREM2 in tau pathology remains unclear thus far. Herein, using P301S mice, we showed that TREM2 was upregulated in microglia during disease progression. Silencing of brain TREM2 exacerbated tau pathology in P301S mice. This exacerbation might be attributed to neuroinflammation-induced hyperactivation of tau kinases. Additionally, more severe neurodegenerative changes and spatial learning deficits were observed following TREM2 silencing. Our results imply that TREM2 attenuates tau kinase activity through restriction of neuroinflammation, and thus protects against tau pathology. These findings further suggest that TREM2 may represent as a potential therapeutic target for tau-related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Objective To elucidate the different roles of lamin A and lamin C in the metastasis of epithelial ovarian cancer (EOC) by examining their expression in EOC tissues and cell lines and their correlations with clinicopathological features. Methods The expression of lamin A and lamin C was assessed in ovarian tissues from 61 EOC patients and 13 normal, 14 benign controls, respectively, using immunohistochemistry. Two pairs of EOC cell lines: HO-8910, HO-8910PM, SKOV-3 and SKOV-3ip were also examined to see the differential expression patterns of lamin A and lamin C. Results Lamin A expression was significantly lower in EOC tissues than that in normal and benign ovarian tissues ( p < 0.05), while lamin C expression was not. Lamin A expression level was closely associated with pathological grades, clinical stages, peritoneal metastasis and lymph node metastasis (all p < 0.05). The progression-free survival rate of the EOC patients with low lamin A expression level was remarkably lower than that of the EOC patients with high expression level ( p < 0.05). Lamin A detected by PCR, Western Blot and immunocytochemistry also showed a significantly lower expression level in HO-8910PM than that in HO-8910 ( p < 0.05), but not for lamin C. There was no difference between SKOV-3 and SKOV-3ip cells. Conclusions The results suggested that loss of lamin A but not lamin C expression in EOC was related to metastasis and poor prognosis. Lamin A may play a critical role in the metastasis of EOC. [ABSTRACT FROM AUTHOR]

Highlights: [•] The phzM overexpression strain showed higher electrochemical activity. [•] Overexpressing phzM decreased the charge transfer resistance of MFC. [•] The electron shuttle (pyocyanin) concentration increased upon phzM overexpression. [•] Genetic modification is efficient to improve the electricity power output of MFC. [ABSTRACT FROM AUTHOR]

Abstract: Cationic polymer PEI-CyD is doped with Nd by plasma technology to produce the gene vector: Nd@PEI-CyD. Luciferase expression and EGFP transfection experiments performed in vitro reveal that Nd@PEI-CyD has significantly higher transfection efficiency than lipofectamine 2000 and PEI-CyD and the mechanism is studied and proposed. The rare-earth element, Nd, stimulates the energy metabolism of cells, enhances cell uptake of complexes/pDNA, and regulates the cellular pathways. These special features suggest a new strategy involving metal-incorporated non-viral gene vectors. [Copyright &y& Elsevier]

Abstract: Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson''s disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function. [Copyright &y& Elsevier]

Abstract: To elucidate the mechanism underlying the testicular effects of 1,1-dichloro-2,2bis(p-chlorophenyl)ethylene (p,p′-DDE), the expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor mRNA and proteins were measured in vivo and in vitro. Sprague-Dawley rats were dosed with p,p′-DDE at 0, 20, 60 and 100mg/kg every other day by intraperitoneal injection for 10 days, and Sertoli cells were treated with p,p′-DDE (0, 10, 30, and 50μM) for 24h. Results indicated that the survival rate of Sertoli cells was decreased with increasing doses of p,p′-DDE. In vitro and in vivo studies, p,p′-DDE could increase the expression of neural cadherin, follicle-stimulating hormone receptor mRNA, while decrease the levels of vimentin, neural cadherin and follicle-stimulating hormone receptor proteins. Moreover, immunohistochemistry analysis revealed that the protein expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor in pubertal rat testes were disrupted by treatment with p,p′-DDE. Taken together, these results suggested that p,p′-DDE exposure could induce testicular toxicity through the changes of the mRNA and protein expressions of vimentin, neural cadherin and follicle-stimulating hormone receptor in vitro and in vivo. [Copyright &y& Elsevier]