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Lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), causes high mortality around the world. Previous studies have suggested that the metabolic pattern of tumor is associated with tumor response to immunotherapy and patient’s survival outcome. Yet, this relationship in LUAD is still unknown. Therefore, in this study, we identified the immune landscape in different tumor subtypes classified by metabolism-related genes expression with a large-scale dataset (tumor samples, n = 2181; normal samples, n = 419). We comprehensively correlated metabolism-related phenotypes with diverse clinicopathologic characteristics, genomic features, and immunotherapeutic efficacy in LUAD patients. And we confirmed tumors with activated lipid metabolism tend to have higher immunocytes infiltration and better response to checkpoint immunotherapy. This work highlights the connection between the metabolic pattern of tumor and tumor immune infiltration in LUAD. A scoring system based on metabolism-related gene expression is not only able to predict prognosis of patient with LUAD but also applied to pan-cancer. LUAD response to checkpoint immunotherapy can also be predicted by this scoring system. This work revealed the significant connection between metabolic pattern of tumor and tumor immune infiltration, regulating LUAD patients’ response to immunotherapy.

Prostate adenocarcinoma (PRAD) is the highest incidence rate of male urogenital morbidity worldwide. Long non-coding RNAs (lncRNAs), as a significant class of gene expression regulators, play a critical role in immune regulation. The purpose of this study is to explore a new immune related lncRNA signature to exactly predict the prognosis of PRAD patients. In this study, we conducted a genome-wide comparative analysis of lncRNA expression profiles in 532 patients with PRAD from the Cancer Genome Atlas (TCGA) database. The immune-related lncRNAs were identified by Cox regression model, and then a new five immune-related lncRNAs signature (FRMD6-AS2, AC008770.3, AC109460.3, AC011899.2, and AC008063.1) were constructed, which could predict the prognosis of PRAD patients. Univariate and multivariate Cox regression analysis showed that the signature could be an independent prognostic indicator of overall survival (OS). Through further study of different clinic-pathological parameters, we found that PRAD samples can be divided into high-risk groups with shorter OS and low-risk groups with longer OS by the signature. Principal component analysis showed that five immune-related lncRNA signature could distinguish the high-risk group from low-risk group in view of the immune-related lncRNAs. The difference of immune status between the two groups was observed by gene set enrichment analysis and the ESTIMATE algorithm. Except FRMD6-AS2, the expression of the other 4 lncRNAs were remarkably up-regulated in tumor tissues. In conclusion, the identified five immune-related lncRNAs signature had important clinical significance in prognosis prediction, and can be used as potential immunotherapy targets for PRAD patients.

Emerging suggest that microRNAs (miRNAs) play vital roles in the occurrence and development of hepatitis B virus (HBV) infectious disease. However, miRNAs in occult hepatitis B virus infection (OBI), a special stage of HBV infection, remain largely unknown. Herein, we conducted this study to identify differentially expressed miRNAs and then to investigate the potential roles of these miRNAs in OBI. Plasma miRNA expression profiles of three OBI patients and three healthy controls were analyzed with high through-put miRNA sequencing technology. Altered expression of miRNAs was further confirmed with reverse transcription quantitative polymerase chain reaction (qRT-PCR). Finally, bioinformatics analysis was conducted to investigate the involved pathways and target genes for these differentially expressed miRNAs. Totally, 32 differentially expressed miRNAs were identified between OBI and healthy controls by miRNA sequencing (fold change ≥ 1.5, P < .1, and counts per million reads ≥ 1), including 16 downregulated and 16 upregulated miRNAs. Differential expression of hsa-miR-486-5p, -25-3p, and -92a-3p and -1-3p was further validated by qRT-PCR analysis, which was consistent with miRNA sequencing analysis. Moreover, these four miRNAs might distinguish OBI from HCs efficiently. Bioinformatics analyses indicated that the differentially expressed miRNAs were primarily involved in various biological processes related to gene expression and transcription, cell development and metabolism, protein modification and kinase activity regulation, as well as multiple signaling pathways such as PI3K/Akt signaling pathway. This study provided a global view of miRNA expression in plasma from OBI patients. These differentially expressed miRNAs might play important roles in the development of OBI, which provided intriguing insights into the screening and molecular mechanism of OBI.

Background DNA methylation (DNAm) is a critical regulator of both development and cellular identity and shows unique patterns in neurons. To better characterize maturational changes in DNAm patterns in these cells, we profile the DNAm landscape at single-base resolution across the first two decades of human neocortical development in NeuN+ neurons using whole-genome bisulfite sequencing and compare them to non-neurons (primarily glia) and prenatal homogenate cortex. Results We show that DNAm changes more dramatically during the first 5 years of postnatal life than during the entire remaining period. We further refine global patterns of increasingly divergent neuronal CpG and CpH methylation (mCpG and mCpH) into six developmental trajectories and find that in contrast to genome-wide patterns, neighboring mCpG and mCpH levels within these regions are highly correlated. We integrate paired RNA-seq data and identify putative regulation of hundreds of transcripts and their splicing events exclusively by mCpH levels, independently from mCpG levels, across this period. We finally explore the relationship between DNAm patterns and development of brain-related phenotypes and find enriched heritability for many phenotypes within identified DNAm features. Conclusions By profiling DNAm changes in NeuN-sorted neurons over the span of human cortical development, we identify novel, dynamic regions of DNAm that would be masked in homogenate DNAm data; expand on the relationship between CpG methylation, CpH methylation, and gene expression; and find enrichment particularly for neuropsychiatric diseases in genomic regions with cell type-specific, developmentally dynamic DNAm patterns.

This research assessed the gene expression patterns related to the synthesis of milk in yak, which is characterized by high fat and protein content but low yield. The yak (Bos grunniens) is one of the most crucial domestic animals in Tibetan life; however, the genetic and molecular factors underlying yak milk protein synthesis remain understudied. Yak mammary biopsies harvested during late-pregnancy (d -15) through the end of subsequent lactation (d 1, 15, 30, 60, 180, and 240) were used to evaluate gene expression via real-time quantitative PCR. The expression pattern of 41 genes encompassing multiple pathways integral to milk protein synthesis including insulin, mammalian target of rapamycin (mTOR), 5' AMP-activated protein kinase, Jak2-Stat5 signaling, and the expression of glucose and AA transporters was evaluated. Our results confirmed that most upregulated genes increased from d -15 and peaked at d 30 or 60 and then remained relatively highly expressed. Specifically, there was an increased expression of mTOR-related amino acid transporters (SLC1A5, SLC7A5, and SLC36A1), glucose transporters (SLC2A1, SLC2A3, and SLC2A8), Jak2-Stat5 pathway (ELF5), and insulin signaling pathway components (IRS1, PDPK1, and AKT1). For activation of proteins synthesis, MTOR was significantly increased only at d 1. Among inhibitors of mTOR signaling, TSC1 and PRKAA2 were significantly upregulated during lactation. The RPL23 was downregulated among ribosomal components. In conclusion, a critical role for AA and glucose transporters and insulin signaling through mTOR for regulation of yak milk protein synthesis was revealed in this study of the yak mammary gland.

BackgroundClear cell renal cell carcinoma (ccRCC) is a urinary disease with high incidence. The high incidence of metastasis is the leading cause of death in patients with ccRCC. This study was aimed to identify the gene signatures during the metastasis of ccRCC.MethodsTwo datasets, including one gene expression profile dataset and one microRNA (miRNA) expression profile dataset, were downloaded from Gene Expression Omnibus (GEO) database. The integrated bioinformatics analysis was performed using the (limma) R package, miRWalk, DAVID, STRING, Kaplan-Meier plotter databases. Quantitative real-time polymerase chain reaction (qPCR) was conducted to validate the expression of differentially expressed genes (DEGs) and DE-miRNAs.ResultsIn total, 84 DEGs (68 up-regulated and 16 down-regulated) and 41 DE-miRNAs (24 up-regulated and 17 down-regulated) were screened from GSE22541 and GSE37989 datasets, respectively. Furthermore, 11 hub genes and 3 key miRNAs were identified from the PPI network, including FBLN1, THBS2, SCGB1A1, NKX2-1, COL11A1, DCN, LUM, COL1A1, COL6A3, SFTPC, SFTPB, miR-328, miR-502, and miR-504. The qPCR data showed that most of the selected genes and miRNAs were consistent with that in our integrated analysis. A novel mRNA-miRNA network, SFTPB-miR-328-miR-502-miR-504-NKX2-1 was found in metastatic ccRCC after the combination of data from expression, survival analysis, and experiment validation.ConclusionIn conclusion, key candidate genes and miRNAs were identified and a novel mRNA-miRNA network was constructed in ccRCC metastasis using integrated bioinformatics analysis and qPCR validation, which might be utilized as diagnostic biomarkers and molecular targets of metastatic ccRCC.

Objective. To identify the key genes involved in prostate cancer and their regulatory network. Methods. The dataset of mRNA/miRNA transcriptome sequencing was downloaded from The Cancer Genome Atlas/the Gene Expression Omnibus database for analysis. The “edgeR” package in the R environment was used to normalize and analyze differentially expressed genes (DEGs) and miRNAs (DEmiRNAs). First, the PANTHER online tool was used to analyze the function enrichment of DEGs. Next, a protein-protein interaction (PPI) network was constructed using STRING and Cytoscape tools. Finally, miRNA-gene regulatory networks were constructed using the miRTarBase. Results. We identified 4339 important DEGs, of which 2145 were upregulated (Up-DEGs) and 2194 were downregulated (Down-DEGs). Functional enrichment analysis showed that the Up-DEGs were related to the immune system and the cell cycle in prostate cancer, whereas the Down-DEGs were related to the nucleic acid metabolic process and metabolism pathways. Twelve core protein clusters were found in the PPI network. Further, the constructed miRNA-gene interaction network showed that 11 downregulated miRNAs regulated 16 Up-DEGs and 22 upregulated miRNAs regulated 22 Down-DEGs. Conclusion. We identified 4339 genes and 70 miRNAs that may be involved in immune response, cell cycle, and other key pathways of the prostate cancer regulatory network. Genes such as BUB1B, ANX1A1, F5, HTR4, and MUC4 can be used as biomarkers to assist in the diagnosis and prognosis of prostate cancer.

Background The effects of C-type natriuretic peptide (CNP) and fibroblast growth factor (FGF)-23 appear to oppose each other during the process of bone formation, whereas few studies exist on the interaction between CNP and FGF-23. The main objective of the present study is to probe whether CNP is directly responsible for the regulation of osteoblast or via antagonizing FGF-23. Methods Osteoblasts were cultured in the absence or presence of CNP (0, 10, and 100 pmol/L) for 24 h, 48 h and 72 h, respectively. Results The findings of the present study indicated that: (1) CNP significantly stimulated osteoblastic proliferation and collagen (Col)-X expression; (2) both osteoblastic (osteocalcin, procollagen type I carboxy-terminal propeptide, total alkaline phosphatase and bone-specific alkaline phosphatase) and osteolytic (tartrate-resistant acid phosphatase and cross-linked carboxyterminal telopeptide of type I collagen) bone turnover biomarkers were up-regulated by CNP in osteoblasts; (3) FGF-23 mRNA and protein were significantly down-regulated at 24 h by CNP in osteoblasts, but the expression of FGF receptor-1/Klotho had no significant change. Conclusions CNP stimulates osteoblastic proliferation and Col-X expression via the down-regulation of FGF-23 possibly in vitro. However, the specific mechanisms of the interaction between CNP and FGF-23 in osteoblasts are still unclear according to our findings. A further study on osteoblasts cultured with CNP and FGF-23 inhibitor will be undertaken in our laboratory.

Background Helios has been reported to stabilize regulatory T (Treg) suppressive function. Programmed cell death protein 1 (PD-1) expression in three human monocyte subsets modulates immune responses. Recently, our team reported that three monocyte subsets are associated with T helper cell differentiation in HIV-1-infected patients. Until now, the effects of monocyte subsets and their PD-1 expression on Foxp3+Helios+ Treg cells have not been fully characterized, especially during acute HIV-1 infection. Results The frequency of Foxp3+Helios+CD45RA+ Treg cells is significantly higher in patients with acute HIV-1 infection than those of healthy controls and chronic HIV-1-infected patients undergoing combined antiretroviral therapy. The frequency of Foxp3+Helios+CD45RA+ Treg cells is inversely correlated with CD4 T-cell counts and the CD4/CD8 ratio in chronic HIV-1-infected patients. During acute HIV-1 infection, the frequency of Foxp3+Helios+CD45RA+ Treg cells is inversely correlated with the frequency of the intermediate CD14++CD16+ monocyte subset, but positively correlated with PD-1 expression in both intermediate CD14++CD16+ and non-classical CD14+CD16++ monocyte subsets. Conclusions In this study, the perturbations of Foxp3+Helios+ Treg cells were characterized, and the association between monocyte subsets and their PD-1 expression and Foxp3+Helios+ Treg cells was evaluated during HIV-1 infection. Our observations provide new evidence of the roles for Foxp3+Helios+ Treg cells and PD-1 expression on monocyte subsets in HIV pathogenesis.

Background Numerous studies have indicated that miRNAs might play significant roles in the development of hepatitis B virus (HBV) infection. while the miRNAs in occult HBV infection (OBI) are still largely unknown. Methods Initially, 15 HBV infection-related miRNAs in plasma of 10 OBI and 10 healthy controls (HCs) was analyzed by qRT-PCR. Significantly dysregulated miRNAs were subsequently validated in another 64 OBI, 20HCs, 31 chronic hepatitis B (CHB) and 20 asymptomatic HBsAg carriers (ASC). Furthermore, the potential biological functions and molecular mechanisms of miR-451a in HBV infection were investigated using HBV-expressing hepatoma cell lines. Results Compared to HCs, plasma miR-451a and miR-340-3p were significantly up-regulated in OBI, ASC and CHB patients, while no significant difference was found among OBI, ASC and CHB patients. ROC curve analysis indicated that both plasma miR-451a and miR-340-3p could moderately distinguish OBI from HCs, with AUCs of 0.76 and 0.78, respectively. When combined, the differentiation efficiency of this miRNA panel was better, with an AUC of 0.82. While, they both could not specifically separate the stage of chronic HBV infection. Functional experiments showed that overexpression of miR-451a might suppress HBV replication and gene expression in hepatoma cell lines. Mechanistically, miR-451a might inhibit HBV replication and gene expression by directly targeting ATF2. Conclusions A plasma panel, including miR-340-3p and miR-451a that might suppress HBV replication by targeting ATF2, has the potential as biomarkers for HBV infection. In the setting of blood donations, this panel would be more practical to moderately differentiate OBI in HBsAg-negative donors.

Ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for bacterial colonization and survival within the gastric mucosa. H. pylori Fur (HpFur) is unique in its ability to regulate gene expression in both metal-bound (holo-Fur) and metal-free (apo-Fur) forms. Bismuth-based drugs are widely used for the treatment of H. pylori infection. However, the mechanism of action of bismuth drug was not fully understood. Recently, it has been reported that bismuth drugs could interfere with the bacterial ferric uptake pathway and inhibit bacterial growth, implying intrinsic correlation between bismuth drug and bacterial iron metabolism. Herein, we demonstrate that Bi(III) binds to HpFur protein specifically at the physiologically important S1 site, which further leads to protein oligomerization and loss of DNA binding capability. The targeting of HpFur by bismuth drugs significantly reduced transcription levels of its regulated genes, which are crucial for bacterial physiology and metabolism. Our studies present direct evidence that perturbation of iron metabolism in H. pylori by bismuth might serve as one of the mechanisms for the antimicrobial activity of bismuth drugs.

PBMCs are essential for immunity and involved in various diseases. To identify genetic variations contributing to PBMCs transcriptome-wide gene expression, we performed a genome-wide eQTL analysis by using genome-wide SNPs data and transcriptome-wide mRNA expression data. To assess whether there are common regulation patterns shared among different tissues/organs, public datasets were utilized to identify common eQTLs shared with PBMCs in lymphoblastoid, monocytes, liver, and brain. Allelic expression imbalance (AEI) assay was employed to validate representative eQTLs identified. We identified 443 cis- and 2386 trans-eSNPs (FDR

MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3′-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA–mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA–mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA–mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA–mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (−0.63–0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced fromNilaparvata lugens, namedNlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), namedYsEF-1αandYsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation forEF-1αthan forEF-1γ. NlEF-1αhas two identical copies. The deduced amino acid sequence homology of NlEF-1αand NlEF-1γis 96 and 64%, respectively, compared withHomalodisca vitripennisandLocusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared withMetarhizium anisopliaeandOphiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT–qPCR) analysis revealed that the expression level ofNlEF-1αandNlEF-1γmRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue.YsEF-1αandYsEF-1γwas highly expressed in fat body. The expression level ofNlEF-1αwas higher than that ofNlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT–qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected withNlEF-1αandNlEF-1γdouble-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype ofN. lugensinjected withYsEF-1αdsRNA was the same as that injected withNlEF-1αdsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences betweenNlEF-1αandYsEF-1α.We demonstrated that partial silencing ofNlEF-1αandNlEF-1γgenes caused lethal and sterility effect onN. lugens.NlEF-1γshares low identity with that of other insects and therefore it could be a potential target for RNAi-based pest management.

Copper nanoparticles (Cu-NPs) have been used increasingly in various products and applications. Although recent studies have reported that exposure to Cu-NPs leads to organ accumulation and obvious toxicity, it remains unclear whether Cu-NPs can be translocated to and cause damage in the uterus. In this study, we investigated the potential for uterine injury and gene expression patterns in female rats exposed to 3.12, 6.25, or 12.5 mg/kg/d Cu-NPs via intraperitoneal injection for 14 consecutive days. The results indicated that exposure to Cu-NPs led to significant decreases in the relative uterine weight coefficients and increases in inflammatory cell infiltration, mitochondrial swelling and vacuolization, shortened and reduced endometrial epithelial cell microvilli, and apoptosis. Furthermore, exposure to Cu-NPs increased malondialdehyde (MDA) accumulation and decreased superoxide dismutase (SOD) levels. Signal transduction mechanism studies indicated that exposure to Cu-NPs activated caspases 3, 8, and 9 and BH3 interacting domain death agonist (tBid), reduced B cell leukemia/lymphoma 2 (Bcl-2) expression, and increased the expression of apoptotic peptidase activating factor 1 (Apaf-1), BCL2-associated X, apoptosis regulator (Bax), and cytochrome c. A microarray analysis revealed significant alterations in the expression of 963 genes; of these, 622 were upregulated and 341 were downregulated. The results of further evaluations of some altered genes, including matrix metallopeptidase 12 (Mmp12), using quantitative RT-PCR agreed with the microarray findings. These results provide strong evidence that Cu-NPs can trigger both intrinsic and extrinsic apoptotic pathways to mediate uterine injury, resulting in oxidative stress-related changes in gene expression.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Currently, the clinical strategies available for the treatment of HCC remain insufficient for the poor prognosis. Sodium/iodide symporter (NIS)-based radioiodine therapy is proposed as a promising therapeutic strategy for the treatment of HCC. However, it is difficult for HCC cells to trap iodine for the lower expression of NIS. Small activating RNA (saRNA) is a newly identified small double-stranded RNA (dsRNA) that can induce endogenous gene expression by targeting promoter sequences. Here, we designed an saRNA (saRNA-482) that targeted the NIS promoter sequences. In the cultured HepG2 cells and Hep3B cells, the expressions of NIS were upregulated after transfection of saRNA-482. In addition, the uptake of 125I increased in the cultured HepG2 and Hep3B cells transfected with saRNA-482. Furthermore, the cell viabilities were significantly inhibited in the saRNA-482-transfected HepG2 and Hep3B cells after 131I treatment. Meanwhile, the apoptosis of saRNA-482-transfected HepG2 and Hep3B cells significantly increased after 131I treatment. The results suggest that RNA activation-mediated upregulation of NIS may have an endoradiotherapeutic potential in the treatment of HCC.

To explore the biological function of rhoptry protein 38 (ROP38) ofThe ROP38 was amplified by RT-PCR fromSDS-PAGE showed that rROP38 was efficient expression with a molecular weight of about 43 kD. Western blot showed that rROP38 reacted with antibody of His tag or human positive antibody, which indicated that ROP38 had good reactogenicity and could be a serological diagnostic antigen.The study successfully obtains the rROP38 of

The brown planthopper Nilaparvata lugens Stal (Homoptera: Delphacidae) can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens , including two different treatments (resistant or susceptible rice) and three virulent N. lugens populations. Three software programs (BestKeeper, Normfinder and geNorm) were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, β-ACT, β-TUB and α-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and α-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and α-TUB were the most suitable reference genes in N. lugens . These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.

Objective To investigate the effect of diabetes with and without vitamin E treatment on testicular metallothionein (MT) and metal (zinc, copper and iron) changes. Methods Diabetes was induced with a single intraperitoneal injection (i.p.) of streptozotocin in rats, and diabetic rats were given Vitamin E by i.p. every other day for 4 weeks. MT protein was measured by the cadmium-haeme assay and metal levels were detected by an atomic absorption spectrophotometer. Results Diabetes did not change testicular MT protein, but significantly increased hepatic MT protein. Diabetes significantly decreased testicular copper, but not hepatic copper. Zinc and iron levels were unchanged in both diabetic testis and liver. Vitamin E significantly enhanced both testicular and hepatic MT, and zinc levels in diabetic rats. Vitamin E slightly decreased the copper levels, but did not change the testicular and hepatic iron in diabetic rats. Conclusions Testicular MT protein expression was not increased, even though hepatic MT significantly increased independent of metal changes, in diabetic rats. Vitamin E enhanced testicular and hepatic MT, which correlated with increased zinc levels.

Aims MicroRNA served as inhibitor for gene expression in various cancers. This study aimed to investigate the role of miR-605 and EN2 in prostate cancer (PCa). Materials and methods In this research, the expression of miR-605 and EN2 protein in PCa tissues and cells were determined by qRT-PCR and western blot, respectively. The cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and the tumor cell invasion assay was accomplished with transwell system. Flow cytometry was used to analyze the cell cycle. The endogenous expression of miR-605 and EN2 was modulated by recombinant plasmids and cell transfection. Dual luciferase reporter assay was performed to determine the interaction between miR-605 and EN2 in PCa cells. Key findings The expression of miR-605 was lower in PCa tissue and cells than that in normal tissues and cells, while the expression of EN2 was just the opposite. Down-regulation of the EN2 by siRNA inhibited the proliferation and invasion of PC3 cells, and the cell cycle was arrested in G0/G1 phase. EN2 regulated the expression of E-cadherin and Vimentin through Snail and EN2 regulated the cell cycle and cell proliferation via PI3K/AKT pathway. MiR-605 inhibited the proliferation and invasion of PCa cells through targeting EN2. Significance EN2 is negatively regulated by miR-605, and down-regulation of miR-605 promotes the proliferation and invasion of PCa cells by up-regulating EN2, which leads to PCa development and progression.

The African oil palm (Elaeis guineensis), which is grown in tropical and subtropical regions, is a highly productive oil-bearing crop. For gene expression-based analyses such as reverse transcription-quantitative real time PCR (RT-qPCR), reference genes are essential to provide a baseline with which to quantify relative gene expression. Normalization using reliable reference genes is critical in correctly interpreting expression data from RT-qPCR. In order to identify suitable reference genes in African oil palm, 17 transcriptomes of different tissues obtained from NCBI were systematically assessed for gene expression variation. In total, 53 putative candidate reference genes with coefficient of variation values

Reverse transcription quantitative real-time polymerase chain reaction (PCR) is a widely used and reproducible method for studying gene expression changes. However, its accuracy and reliability is highly dependent on the normalization step. Cocos nucifera L., a perennial palm with a long productive life span, is frequently exposed to soil and atmospheric drought and other stresses. In applying gene expression analysis to understand coconut stress responses, validation of suitable reference genes is an important first step. In this study, seven putative reference genes were identified from coconut transcriptome data. The stability of these putative reference genes was assessed in a diverse set of 18 coconut samples subject to cold, drought, and high-salinity treatment and representing different endosperm developmental stages. Using statistical algorithms geNorm, NormFinder, and BestKeeper, eEF1-α and UBC10 genes were identified as stable reference genes in all stress treatments and endosperm development stages, consistent with validated reference genes in rice (Oryza sativa L.), potato (Solanum tuberosum L.), and other species. Further validation of analyzed reference genes by normalization of a C-repeat binding factor (CBF)-like gene in cold-treatment samples indicated that the less stable reference genes produced different results from the more stable reference genes, with weaker variation or higher expression levels of a target gene. Our results will be beneficial for further research on molecular mechanisms of stress resistance.

Several biomaterials have been introduced for bone augmentation. However, information is lacking about the mechanisms of bone regeneration and/or integration of these materials in the recipient bone. This study aimed to determine the molecular and structural events in bone defects after augmentation with synthetic tetrapod-shaped calcium phosphate (Tetrabone; TetraB) compared with natural deproteinized bovine bone (DBB). Defects were created in the epiphyses of rat femurs and filled with TetraB or DBB or left empty (Sham). After 3, 6, 14 and 28 d, samples were harvested for histology, histomorphometry, ultrastructure and gene expression analyses. At 3 d, higher expressions of bone formation (ALP and OC) and remodeling (CatK) genes were detected in TetraB compared with DBB and Sham. Downregulation of bone remodeling genes (TRAP and CatK) was detected in DBB as compared to Sham after 14 d. Histomorphometry at 6 and 14 d demonstrated greater bone contact with the granules in TetraB. At 28 d, a larger bone area per defect was found in TetraB. The present experiments show that a synthetic substitute, consisting of α-tricalcium and octacalcium phosphates, induces early osteogenic and osteoclastic activities and promotes bone formation in trabecular bone defects.

Aim To explore the feasibility of allogeneic mesenchymal stem cells (MSCs) transplanted intravenously for angiogenesis and bone repair in a rabbit model of avascular necrosis of femoral head (ANFH). Materials and methods Forty-five rabbits were randomized into three groups: a blank control group (without treatment), a necrotic control group (ANFH induced but without therapy), and an MSC transplantation group (ANFH induced and treated with MSC transplantation). The biopsies, blood sampling, and imaging examinations were performed on each animal at different time points (2, 4, and 6 wk). To monitor angiogenesis and bone repair progress, examinations included real-time polymerase chain reaction, Western blot analysis, x-ray, computed tomography, Masson trichrome staining, picrosirius red staining, and immunohistochemical staining. Results Necrosis and bone collapse were observed in bilateral femoral heads of necrotic rabbits of the necrotic control group, whereas the femoral head morphology was generally restored in the MSC transplantation group. The mRNA levels of Cbfa1, BMP, VEGF, and OPN in bone tissue were significantly higher in the MSC transplantation group than in the necrotic control group. In addition, the total protein amount of Cbfa1 in the MSC transplantation group was also significantly higher than that in the necrotic control group (P Conclusion Intravenous transplantation of allogeneic MSCs can promote vascular and bone regeneration in the necrotic region of the femoral head in a rabbit model of ANFH. The results of our study suggest that the intravenous transplantation of MSCs could be a potential and minimally invasive treatment option for ANFH patients.

Background Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a recently identified adipokine. Studies suggest it is involved in many diseases such as obesity, diabetes and coronary artery disease (CAD). This study is to investigate the association of single nucleotide polymorphisms (SNPs) in vaspin with CAD and its potential mechanisms. Methods A total of 1570 consecutive patients undergoing coronary angiography were enrolled and the genotypes were determined by TaqMan allelic discrimination. Serum vaspin concentrations and mRNA expression levels were determined by ELISA and RT-PCR, respectively. Reporter gene assay was performed to investigate the effect of polymorphism on vaspin promoter function. Results After multivariate analysis, allele A of rs2236242 was found as an independent determinant of CAD (OR = 1.32, p = 0.004). Rs35262691 in vaspin promoter was associated with serum vaspin concentration and mRNA expression in peripheral blood mononuclear cells (PBMC) though no association had been found with CAD. Reporter gene assay further confirmed that CC genotype of rs35262691 had 2.1 ± 0.4-fold higher activities than TT genotype in facilitating gene expression. Conclusions Our results show that the variants of vaspin gene are associated with serum vaspin levels and risk for CAD in Chinese population.

Objective Rheumatoid arthritis (RA) is a complex autoimmune disease. Using a gene-based association research strategy, the present study aims to detect unknown susceptibility to RA and to address the ethnic differences in genetic susceptibility to RA between European and Asian populations. Methods Gene-based association analyses were performed with KGG 2.5 by using publicly available large RA datasets (14,361 RA cases and 43,923 controls of European subjects, 4,873 RA cases and 17,642 controls of Asian Subjects). For the newly identified RA-associated genes, gene set enrichment analyses and protein-protein interactions analyses were carried out with DAVID and STRING version 10.0, respectively. Differential expression verification was conducted using 4 GEO datasets. The expression levels of three selected ‘highly verified’ genes were measured by ELISA among our in-house RA cases and controls. Results A total of 221 RA-associated genes were newly identified by gene-based association study, including 71‘overlapped’, 76 ‘European-specific’ and 74 ‘Asian-specific’ genes. Among them, 105 genes had significant differential expressions between RA patients and health controls at least in one dataset, especially for 20 genes including 11 ‘overlapped’ (ABCF1, FLOT1, HLA-F, IER3, TUBB, ZKSCAN4, BTN3A3, HSP90AB1, CUTA, BRD2, HLA-DMA), 5 ‘European-specific’ (PHTF1, RPS18, BAK1, TNFRSF14, SUOX) and 4 ‘Asian-specific’ (RNASET2, HFE, BTN2A2, MAPK13) genes whose differential expressions were significant at least in three datasets. The protein expressions of two selected genes FLOT1 (P value = 1.70E-02) and HLA-DMA (P value = 4.70E-02) in plasma were significantly different in our in-house samples. Conclusion Our study identified 221 novel RA-associated genes and especially highlighted the importance of 20 candidate genes on RA. The results addressed ethnic genetic background differences for RA susceptibility between European and Asian populations and detected a long list of overlapped or ethnic specific RA genes. The study not only greatly increases our understanding of genetic susceptibility to RA, but also provides important insights into the ethno-genetic homogeneity and heterogeneity of RA in both ethnicities.

The current lack of complete understanding of the pathogenesis of acute kidney injury (AKI) is a significant barrier to its early diagnosis and treatment. Cell cycle arrest plays an important role in the protection of renal tubular epithelial cells and maladaptive repair following AKI. G1 phase cell arrest serves as a protective mechanism following AKI, avoiding replication of damaged DNA. Insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinase-2 (TIMP-2) are closely associated with G1 cell cycle arrest during the very early phase of cellular damage and can serve as an ideal biomarker to predict AKI. However, sustained cell cycle arrest after severe AKI may result in cell senescence and maladaptive repair, with typical characteristics of the development of cell cycle arrest in the gap 2 (G2) or mitotic (M) phase. Markers of cell cycle arrest signal and spread the “alarm” from the site of injury to adjacent cells in an autocrine or paracrine manner, giving rise to abnormal amplification and release of profibrogenic factors, activation of pericytes/perivascular fibroblasts, and eventually fibrosis. Therefore, cell cycle regulation has become a potentially new target for the prevention and treatment of AKI. In this review, we summarize the characteristics of the cell cycle following AKI and the markers of cell cycle arrest that enable the early detection of AKI. We also discuss how to prevent the progression of chronic kidney disease (CKD) by regulating cell cycle arrest.

Objective Systemic lupus erythematosus (SLE) is a complex auto-immune disease. Gene expression studies have been conducted to identify SLE-related genes in various types of samples. It is unknown whether there are common marker genes significant for SLE but independent of sample types, which may have potentials for follow-up translational research. The aim of this study is to identify common marker genes across various sample types for SLE. Methods Based on four public microarray gene expression datasets for SLE covering three representative types of blood-born samples (monocyte; peripheral blood mononuclear cell, PBMC; whole blood), we utilized three statistics (fold-change, FC; t-test p value; false discovery rate adjusted p value) to scrutinize genes simultaneously regulated with SLE across various sample types. For common marker genes, we conducted the Gene Ontology enrichment analysis and Protein-Protein Interaction analysis to gain insights into their functions. Results We identified 10 common marker genes associated with SLE (IFI6, IFI27, IFI44L, OAS1, OAS2, EIF2AK2, PLSCR1, STAT1, RNASE2, and GSTO1). Significant up-regulation of IFI6, IFI27, and IFI44L with SLE was observed in all the studied sample types, though the FC was most striking in monocyte, compared with PBMC and whole blood (8.82–251.66 vs. 3.73–74.05 vs. 1.19–1.87). Eight of the above 10 genes, except RNASE2 and GSTO1, interact with each other and with known SLE susceptibility genes, participate in immune response, RNA and protein catabolism, and cell death. Conclusion Our data suggest that there exist common marker genes across various sample types for SLE. The 10 common marker genes, identified herein, deserve follow-up studies to dissert their potentials as diagnostic or therapeutic markers to predict SLE or treatment response.

Perfluorooctane sulfonate (PFOS) could induce neonatal pulmonary injuries in rodents. The aim of this study was to investigate the underlying mode of action. Pregnant rats were dosed orally with PFOS (0, 0.1 and 2.0mg/kgd) from gestation days (GD) 1 to 21. Lung samples from postnatal day (PND) 0 and 21 pups were analyzed for the toxic effects of PFOS. The results showed that maternal exposure to 2.0mg/kgd PFOS caused severe histopathological changes along with marked oxidative injuries and cell apoptosis in offspring lungs; at the same time, the ratio of Bax to Bcl-2, release of cytochrome c (Cyt c) from mitochondria to cytoplasm, expressions of Fas and Fas-L, and activities of caspase-3, -8 and -9 were up-regulated correspondingly. The results indicate that oxidative stress and both intrinsic and extrinsic cell death pathways were involved in prenatal PFOS exposure-induced injuries in postnatal lungs.

Xenobiotics exposure in early life may have adverse effects on animals’ development through mitochondrial injury or dysfunction. The current study demonstrated the possibility of cardiac mitochondrial injury in prenatal PFOS-exposed weaned rat heart. Pregnant Sprague-Dawley (SD) rats were exposed to perfluorooctane sulfonate (PFOS) at doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2–21. The dams were allowed to give nature delivery and then heart tissues from weaned (postnatal day 21) offspring rats were analyzed for mitochondrial injury through ultrastructure observation by electron microscope, global gene expression profile by microarray, as well as related mRNA and proteins expression levels by quantitative PCR and western blot. Ultrastructural analysis revealed significant vacuolization and inner membrane injury occurred at the mitochondria of heart tissues from 2.0 mg/kg/d dosage group. Meanwhile, the global gene expression profile showed significant difference in level of some mRNA expression associated with mitochondrial function at 2.0 mg/kg/d dosage group, compared to the control. Furthermore, dose-response trends for the expression of selected genes were analyzed by quantitative PCR and western blot analysis. The selected genes were mainly focused on those encoding for proteins involved in energy production, control of ion levels, and maintenance of heart function. The down-regulation of mitochondrial ATP synthetase (ATP5E, ATP5I and ATP5O) implicated a decrease in energy supply. This was accompanied by down-regulation of gene transcripts involved in energy consumption such as ion transporting ATPase (ATP1A3 and ATP2B2) and inner membrane protein synthesis (SLC25A3, SLC25A4, SLC25A10, SLC25A29). The up-regulation of gene transcripts encoding for uncoupling proteins (UCP1 and UCP3), epidermal growth factor receptor (EGFR) and connective tissue growth factor (CTGF), was probably a protective process to maintain heart function. The results indicate PFOS prenatal exposure can induce cardiac mitochondrial injury and gene transcript change, which may be a significant mechanism of the developmental toxicity of PFOS to rat.

Perfluorooctane sulfonate (PFOS) is an environmental organic pollutant, the potential neurotoxicity of which is causing great concern in fish. In the present study, we examined the effects of PFOS on motor neurons, and investigated the potential toxicological mechanisms oxidative stress in zebrafish embryos. Six-hour post-fertilization (hpf) zebrafish embryos were exposed to 1.0 mg/L PFOS, then we examined the expression of alpha-tubulin, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 5 (CDK5), and peroxiredoxin 2 (PRX2) after PFOS exposure until 120 hpf. The results showed that PFOS increased alpha-tubulin in the coccygeal spinal cord (CSC) at 96 hpf, whereas decreased alpha-tubulin in the brain and spinal cord at 120 hpf. PCNA expression was highly increased in CSC and abdomen compared with control at 96 and 120 hpf after PFOS exposure. In addition, PFOS exposure caused CDK5 expression to be highly increased in brain region following by down-regulation of PRX2 expression at 96 hpf. These results indicated that, at least in part, the effect on motor neurons induced by PFOS was mediated by dynamically interfering with the expression of alpha-tubulin and PCNA. Furthermore, PFOS-induced toxicity was associated with oxidative stress by deregulating CDK5 and PRX2.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that inhibit gene expression post-transcriptionally. By regulating their target genes, miRNAs play important roles in tumor generation and development. Recently, the mir-200 family was revealed to inhibit the epithelial-mesenchymal transition, which is viewed as an essential step in early tumor metastasis. Here, we used luciferase assays to demonstrate that mir-200b interacts with predicted target sites in the 3' untranslated region of RND3. In HeLa cells, mir-200b directly reduced the expression of RND3 at the mRNA and protein levels, which thereby promoted expression of the downstream protein cyclin D1 and increased S-phase entry. In conclusion, our study demonstrates a novel role for mir-200b in cell cycle progression and identifies RND3 as a novel mir-200b target.

The different expression of type X collagen and Runx2 between the convex and concave side of vertebral growth plate in scoliosis may help to improve our understanding of the role that growth plate tissue play in the development or progression of idiopathic scoliosis. In this investigation, there were significant differences of the total expression of type X collagen, Runx2 protein, and Runx2 mRNA between convex side and concave side growth plates of the apex vertebrae (p0.05). The total expression of type X collagen in the concave side growth plates of the lower end vertebrae was higher than that in the same side growth plates of apex (p0.05). The total expression of Runx2 in the concave side growth plates in the upper and lower end vertebrae were higher than that in the concave side growth plates of apex (p0.05). The expression of type X collagen, Runx2, and Runx2 mRNA, the cell density of type X collagen and Runx2 positive chondrocytes, and histological changes between convex side and concave side of the vertebral growth plate indicated that the vertebral growth plate was affected by mechanical forces, which was a secondary change and could contribute to progression of adolescent idiopathic scoliosis.

UV radiation from the sun impacts skin health adversely through complex, multiple molecular pathways. Premature skin aging (photoaging) is among the most widely appreciated harmful effects of chronic exposure to solar UV radiation. Extensive damage to the dermal connective tissue is a hallmark of photoaged skin. Disruption of the normal architecture of skin connective tissue impairs skin function and causes it to look aged. UV irradiation induces expression of certain members of the matrix metalloproteinase (MMP) family, which degrade collagen and other extracellular matrix proteins that comprise the dermal connective tissue. Although the critical role of MMPs in photoaging is undeniable, important questions remain. This article summarizes our current understanding of the role of MMPs in the photoaging process and presents new data that (1) describe the expression and regulation by UV irradiation of all members of the MMP family in human skin in vivo and (2) quantify the relative contributions of epidermis and dermis to the expression of UV irradiation-induced MMPs in human skin in vivo.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 20-24; doi:10.1038/jidsymp.2009.8.

Myelin basic protein (MBP), as a major component of the myelin sheath, has been revealed to play an important role in forming and maintaining myelin structure in vertebrate nervous system. In teleost, hypothalamus is an instinctive brain center and plays significant roles in many physiological functions, such as energy metabolism, growth, reproduction, and stress response. In comparison with other MBP identified in vertebrates, a smallest MBP is cloned and identified from the orange-spotted grouper hypothalamic cDNA plasmid library in this study. RT-PCR analysis and Western blot detection indicate that the EcMBP is specific to hypothalamus, and expresses mainly in the tuberal hypothalamus in adult grouper. Immunofluorescence localization suggests that EcMBP should be expressed by oligodendrocytes, and the expressing cells should be concentrated in hypothalamus and the area surrounding hypothalamus, such as NPOpc, VC, DP, NLTm, and NDLI. The studies on EcMBP expression pattern and developmental behaviour in the brains of grouper embryos and larvae reveal that the EcMBP-expressing cells are only limited in a defined set of cells on the border of hypothalamus, and suggest that the EcMBP-expressing cells might be a subpopulation of oligodendrocyte progenitor cells. This study not only identifies a smallest MBP isoform specific to hypothalamus that can be used as a molecular marker of oligodendrocytes in fish, but also provides new insights for MBP evolution and cellular distribution.

Background Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-β is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-β expression is stimulated in KKs. Materials and methods Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed usingquantitative real-time polymerase chain reaction. TGF-β activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively. Results Increased transcription of TGF-β2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-β3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-β1. Increased TGF-β2 mRNA correlated with increased TGF-β biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-β2, TβR1, and TβR2 in KKs. Conclusions KKs produce more TGF-β2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-β activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control.