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IntroductionA kidney tumor is among the 10 most common cancers. Among kidney tumors, renal cell carcinoma (RCC) is one of the most common types with an alarming increasing incidence rate. Although the disruption of microbiota is an established factor in the progression of intestinal cancers, its role in other types of cancers has been under-studied.Material and methodsIn this study, the microbiome disruption and the involvement of SNZ (SCHNARCHZAPFEN) and SA (Stromalin) genes in the development of kidney cancer have been focused on using a combination of genetic and bioinformatic analysis. The microbiomes of kidney tumor patients were analyzed using various genetic and bioinformatic variations. Genetic and bioinformatic analyses were performed to identify operational taxonomic units (OTUs), SNZ, SA, and annotate species were determined using 41 samples from a population of kidney tumors.ResultsThe whole samples from the kidney tumor of patients were screened by PCR amplification and a total of 1317 OTUs were identified. Among them, 379 were common among the two populations, 766 were unique to the SA gene, and 172 to SNZ. SA was more abundant in Gammaproteobacteria and bacilli, while SNZ had a higher abundance in bacteroidia and actinobacteria. Correlation analysis was performed to find out the bacteria that were differentially expressed among the population samples.ConclusionsTo sum up, our study reveals that SA and SNZ are differentially expressed in the microbiome of the kidney tumor that is associated with the development of kidney tumors such as renal cell carcinoma in human populations.

Copper (Cu) pollution in water and agricultural soil has always been a worldwide concern. This research aims to investigate the health effects of copper exposure on Caenorhabditis elegans (C. elegans) under the existing environmental quality standards (1 mg/L and 2 mg/L) via lifespan, reproduction, biological markers and transcriptome analysis. The results showed that copper of these two environmental standards shorten the lifespan of nematodes, reduced the brood size, reduced the frequency of pharyngeal pumps and prolonged defecation time as aging-related behaviors, and increased the levels of aging-related markers ROS, MDA and H2O2. There was a certain effect trend for the two exposure concentrations. Further, the possible molecular mechanism of copper-induced aging and reproductive effects on C. elegans was explored. Differential gene expression analysis was performed, and 2332 genes (567 up- and 1765 down-regulated genes) in the 1 mg/L group, 2449 DEGs (724 up- and 1725 down-regulated genes) in the 2 mg/L group in response to copper treatment. The top 20 regulated genes were vit (vit-1, vit-3, vit-4) genes, col genes (col-35, col-72, col-114, col-123, col-164, col-183, col-185), eea-1, him-18 and grl-20, which suggested that cuticle collagen synthesis and yolk expression were disrupted by copper. Analysis of KEGG pathway showed copper exposure widely affects longevity regulation pathways, thereby promoting aging. In summary, the sequencing results extensively and deeply reveal the health hazards of environmentally relevant doses of copper exposure to C. elegans, and behavioral testing verified that copper promoted aging of C. elegans.

Peroxidase 3 (Prx‐3) is an important antioxidant protein that protects the body from the toxic effects of reactive oxygen species. In this study, the full‐length Prx3 cDNA sequence (PmPrx3) was isolated from Penaeus monodon. The PmPrx3 cDNA was 942 base pairs long and encoded a 227 amino acid polypeptide. The PmPrx3 protein showed high similarity (74%–83%) with the Prx3 protein from other species and contained the conserved Prx domain and peroxidase catalytic center. The results of reverse transcription polymerase chain reaction showed that PmPrx3 mRNA was universally expressed in all tested tissues. The level of PmPrx3 mRNA expression in the gills and hepatopancreas significantly changed after heavy metal exposure. Recombinant PmPrx3 protein (rPmPrx3) was obtained using a prokaryotic expression system and purified using affinity chromatography. Antioxidant activity assays showed that rPmPrx3 reduced insulin in the presence of dithiothreitol, suggesting that the anti‐oxidant function of rPmPrx3 was thiol‐dependent. In addition, Escherichia coli transferred with pET‐28a‐PmPrx3 was significantly protected in response to metal toxicity and H₂O₂ oxidative stress. In summary, our findings provided important insights into the mechanism of action of the Prx3 gene in environmental stress in P. monodon.

Exposure to airborne particulate matter (PM) 2.5 induced various adverse health effects, such as metabolic syndrome, systemic inflammation and respiratory infection. However, a global influence of PM2.5-induced metabolic and proteomic disorders remains confusing, and the underlying mechanism is still under-explored. Herein, LC-MS/MS-based metabolomics, lipidomics and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics were applied to analyze the toxicological characteristics of PM2.5 from Taiyuan City in China (Taiyuan-PM2.5) on human lung carcinoma cells (A549) after the 24-h treatment. Metabolites, lipids and proteins that have distinctive differences were screened by SIEVE, LipidSearch and Proteome Discoverer, respectively. The abundance of 56 metabolites (40 increased and 16 decreased), 22 lipids (19 increased and 3 decreased) and 81 proteins (55 up-regulated and 26 down-regulated) were significantly changed upon the PM2.5 treatment. Among the proteomics analysis, 16 proteins were specifically related to RNA splicing, mainly including up-regulated serine/arginine-rich splicing factor 1 (SRSF1), SRSF2, small nuclear ribonucleoprotein 70 kDa (snRNP70), small nuclear ribonucleoprotein polypeptide B (SNRPB), SNRPC, SNRPE and down-regulated heterogeneous nuclear ribonucleoprotein U-like 2 (hnRNP UL2). At the metabolic level, PM2.5 exposure significantly altered the sphingolipid metabolism, including ceramide, serine, sphingosine and sphingomyelin. It was proposed that excessive accumulation of ceramide and expression of key enzymes (ceramide synthases, phingomyelinase, sphingosine kinase types 2 and protein phosphatase-1) induced the secretion of pro-inflammatory cytokines, generation of lipotoxicity and alterations of RNA splicing in PM2.5-treated A549 cells. In general, our results demonstrated that ceramide accumulation and altered RNA splicing could becritical contributors to PM2.5-induced cytotoxicity at metabolic and proteomic level, which might be considered as potential markers for toxicological evaluation of PM2.5 samples.

Estuarine environmental have been reported to undergo significant fluctuations in oxygen concentrations with hypoxic conditions and subsequent re-oxygenation events being of significant concern for resident fish populations. In this study we assessed the toxicological effects of hypoxia and re-oxygenation on the liver of hypoxia-sensitive spotted sea bass (Lateolabrax maculatus) that were exposed to hypoxia (1.17 mg/L dissolved oxygen) for 12 h and then re-oxygenated for 12 h. The activities of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in serum significantly increased under hypoxia (p < 0.05) and continued to increase during re-oxygenation (p < 0.05), indicating that normal liver function might be disrupted by hypoxia and might become worse during re-oxygenation for 12h. Total protein, albumin, and globulin levels in serum decreased under hypoxia but began to return to normal during re-oxygenation, showing that protein synthesis in the liver decreased during hypoxia but could be restored by re-oxygenation. We also used RNA-Seq technology to identify changes in gene expression in the liver during hypoxia and re-oxygenation. Transcriptome sequencing revealed that the hypoxia-inducible factor (HIF-1) signaling pathway, apoptosis, and purine metabolism transcripts were significantly enriched under hypoxia and re-oxygenation conditions. A total of 15 and 16 apoptosis-related genes were induced by hypoxia and re-oxygenation stress, respectively. The apoptosis index increased from the normal to the hypoxic condition and was highest under re-oxygenation. Additionally, 19 and 29 genes, that are involved in purine metabolism in the liver of L. maculatus during hypoxia and re-oxygenation, respectively, were dysregulated. Unexpectedly, the serum uric acid level significantly increased during hypoxia and significantly decreased under re-oxygenation, indicating the presence of purine metabolic disorder in the liver of L. maculatus. These results illustrate that hypoxia poses a pronounced threat to hepatocyte function in L. maculatus and that liver damage is difficult to reverse with 12 h of re-oxygenation, and it may actually become worse when re-oxygenation is established.

As a hepadnavirus, hepatitis B virus (HBV) can cause damage to extrahepatic organs. The kidney is one of the organs that is more susceptible to damage. Research studies on HBV-associated glomerulonephritis (HBV-GN) have been going on for decades. However, the underlying molecular mechanism remains obscure. Here, we applied a tandem mass tag (TMT) isobaric labeling-based method to quantitatively profile the kidney proteome of HBV transgenic mice to illustrate the pathological mechanisms of HBV-GN. Weighted correlation network analysis, a clustering method for gene expression, is used to cluster proteins. Totally, we identified 127 proteins that were highly associated with HBV expression out of a total of 5169 quantified proteins. Among them, the downregulated solute carrier (SLC) family proteins are involved in the process of HBV-GN. We also found that IL1B was upregulated in the kidney tissue of HBV transgenic mice. These findings suggest that HBV disrupts the small molecule transport network of the kidney, which contributes to the occurrence of HBV-GN. The transporter, particularly SLC family 7 member 7 (SLC7A7), is involved in this process, which might serve as an intervention target for HBV-GN. All MS data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD016450.

Plenty of evidence showing that microRNAs (miRNAs) post-transcriptionally regulate gene expression and are involved in a wide range of biological processes. However, the roles of miRNAs in ovarian development process remain largely unknown in shrimp. In the present study, high-throughput sequencing of small RNAs was performed to find specific miRNAs that are involved in ovarian development process in Penaeus monodon. Two small RNA libraries were constructed from undeveloped (UNDEV group) and developed (DEV group) ovarian tissues in P. monodon. In total, 43 differentially expressed miRNAs were identified between the two groups (P ≤ 0.05, |log2 ratio| ≥1), and their expression profiles were validated by qRT-PCR. In order to further clarify the functional roles of these differentially expressed miRNAs during ovarian development process, target gene prediction was performed. In total, 4,102 target genes of 43 miRNAs were predicted, then clustered by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database; only four specific pathways related to ovarian development were obtained (P P. monodon.

Xylem development is a key process for wood formation in woody plants. To study the molecular regulatory mechanisms related to xylem development in hybrid poplar P. davidiana × P. bolleana, transcriptome analyses were conducted on developing xylem at six different growth stages within a single growing season. Xylem development and differentially expressed genes in the six time points were selected for a regulatory analysis. Xylem development was observed in stem sections at different growth stages, which showed that xylem development extended from the middle of April to early August and included cell expansion and secondary cell wall biosynthesis. An RNA-seq analysis of six samples with three replicates was performed. After transcriptome assembly and annotation, the differentially expressed genes (DEGs) were identified, and a Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and expression analysis of the DEGs were performed on each sample. On average, we obtained &gt, 20 million clean reads per sample, which were assembled into 84,733 nonredundant transcripts, of which there were 17,603 unigenes with lengths &gt, 1 kb. There were 14,890 genes that were differentially expressed among the six stages. The upregulated DEGs were enriched in GO terms related to cell wall biosynthesis between S1 vs. S2 or S3 vs. S4 and, in GO terms, related to phytohormones in the S1 vs. S2 or S4 vs. S5 comparisons. The downregulated DEGs were enriched in GO terms related to cell wall biosynthesis between S4 vs. S5 or S5 vs. S6 and, in GO terms, related to hormones between S1 vs. S2 or S2 vs. S3. The KEGG pathways in the DEGs related to “phenylpropanoid biosynthesis”, “plant hormone signal transduction” and “starch and sucrose metabolism” were significantly enriched among the different stages. The DEGs related to cell expansion, polysaccharide metabolism and synthesis, lignin synthesis, transcription factors and hormones were identified. The identification of genes involved in the regulation of xylem development will increase our understanding of the molecular regulation of wood formation in trees and, also, offers potential targets for genetic manipulation to improve the properties of wood.

Osteoarthritis (OA) is characterized by degradation of chondrocyte extracellular matrix (ECM). Accumulating evidence suggests that microRNAs (miRNAs) are associated with OA, but little is known of their function in chondrocyte ECM degradation. The objective of this study was to investigate the expression and function of miRNAs in OA. miRNA expression profile was determined in OA cartilage tissues and controls, employing Solexa sequencing and reverse transcription quantitative PCR (RT-qPCR). According to a modified Mankin scale, cartilage degradation was evaluated. Functional analysis of the miRNAs on chondrocyte ECM degradation was performed after miRNA transfection and IL-1β treatment. Luciferase reporter assays and western blotting were employed to determine miRNA targets. Expression of miR-221-3p was downregulated in OA cartilage tissues, which was significantly correlated with a modified Mankin scale. Through gain-of-function and loss-of-function studies, miR-221-3p was shown to significantly affect matrix synthesis gene expression and chondrocyte proliferation and apoptosis. Using SW1353 and C28I2 cells, SDF1 was identified as a target of miR-221-3p. SDF1 overexpression resulted in increased expression of catabolic genes such as MMP-13 and ADAMTS-5 in response to IL-1β, but these effects were moderated by miR-221-3p. SDF1 treatment antagonized this effect, while knockdown of SDF1 by shSDF1 induced inhibitory effects on the expression of CXCR4 and its main target genes, similar to miR-221-3p. The results indicate that upregulation of miR-221-3p could prevent IL-1β-induced ECM degradation in chondrocytes. Targeting the SDF1/CXCR4 signaling pathway may be used as a therapeutic approach for OA. miR-221-3p is downregulated in human cartilage tissues. miR-221-3p levels are associated with cartilage degeneration grade. miR-221-3p upregulation prevents IL-1β-induced ECM degradation in chondrocytes. Protection of ECM degradation by miR-223-3p occurs via SDF1/CXCR4 signaling. miR-221-3p is identified as a novel potential therapeutic target for osteoarthritis. KEY MESSAGES: miR-221-3p is downregulated in human cartilage tissues. miR-221-3p levels are associated with cartilage degeneration grade. miR-221-3p upregulation prevents IL-1β-induced ECM degradation in chondrocytes. Protection of ECM degradation by miR-223-3p occurs via SDF1/CXCR4 signaling. miR-221-3p is identified as a novel potential therapeutic target for osteoarthritis.

Current studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) are closely related in acute myocardial infarction (AMI). Previous studies have shown that miR-125b promotes fibrosis and up-regulates in cardiac fibroblasts (CFs), and further experiments showed that circ_LAS1L had multiple binding sites of miR-125b, and their expression was inversely related in AMI patients and CFs. RNA immunoprecipitation (RIP), pull down, and dual luciferase reporter gene assay all confirmed that miR-125b directly bound to circ_LAS1L. Circ_LAS1L overexpression promoted the expression of downstream target gene secreted frizzled-related protein 5 (SFRP5), inhibited the expression of alpha-SMA, collagen I, and collagen III, inhibited CF proliferation and migration, and promoted apoptosis. When cotransfected with circ_LAS1L overexpression vector and miR-125b mimics, the above gene expression and CF biological behaviours did not change significantly. But when cotransfected with circ_LAS1L overexpression vector and SFRP5 siRNA, SFRP5 expression was still down-regulated, the expression of alpha-SMA, collagen I, and collagen III was up-regulated, and the proliferation and migration of CFs were increased. Therefore, circ_LAS1L inhibits the activity of miR-125b by adsorbing it, thus promoting the expression of SFRP5 and then regulating the biological characteristics of CFs. These findings may provide an important experimental basis for the regulation of myocardial fibrosis after myocardial infarction. SIGNIFICANCE OF THE STUDY: Studies have shown that circular RNAs (circRNAs) play important roles in cardiovascular diseases, but there are few studies on their roles in the process of myocardial fibrosis. In this study, we found that circ_LAS1L was down-regulated in acute myocardial infarction (AMI) patients and cardiac fibroblasts (CFs), and could bind directly to miR-125b, thereby promoting the expression of downstream target gene secreted frizzled-related protein 5 (SFRP5), ultimately inhibiting the activation, proliferation and migration of CF, and promoting apoptosis. This suggests that circ_LAS1L/miR-125b/SFRP5 pathway can regulate the biological function of CF and may play an important role in the process of myocardial fibrosis, thus providing an important theoretical basis for the regulation of myocardial fibrosis after myocardial infarction.

Brown seaweed Sargassum confusum (C. Agardh) has been used in traditional Chinese medicine to treat a variety of diseases. The aim of the present study was to evaluate the anti-diabetic effect of oligosaccharides from brown seaweed S. confusum (SCO). The anti-diabetic effect of SCO was evaluated in vivo using high-fat/high-sucrose fed hamsters. Molecular mechanisms of modulating gene expression of specific members of insulin signaling pathways were determined. The components of the intestinal microflora in diabetic animals were also analyzed by high-throughput 16S rRNA gene sequencing. And it was found that SCO had a sequence of sulfated anhydrogalactose and methyl sulfated galactoside units. Fasting blood glucose levels were significantly decreased after SCO administration. Histology showed that SCO could protect the cellular architecture of the liver. SCO could also significantly increase the relative abundance of Lactobacillus and Clostridium XIVa and decrease that of Allobaculum, Bacteroides and Clostridium IV. The active role of SCO in anti-diabetic effect was revealed by its regulation of insulin receptor substrate 1/phosphatidylinositol 3-kinase and c-Jun N-terminal kinase pathways. These results suggested that SCO might be used as a functional material to regulate gut microbiota in obese and diabetic individuals.

The α,β-unsaturated-enone contained natural products have been reported showing NF-κB inhibition effect. It is well known that NF-κB inhibitors can also be used to inhibit osteoclastogenesis. In a continual discovery new agents for anti-osteoclastogenesis, 8 different type compounds with α,β-unsaturated-enone fragments from our in-house library were evaluated for NF-κB inhibition and anti-osteoclastogenesis. Experimental results indicated five compounds exhibited inhibition of NF-κB signal pathway. Among them, one compound ((E)-2-(4-fluorobenzylidene)-3,4-dihydronaphthalen-1(2H)-one, 6a) simultaneously inhibits both osteoclastogenesis and NF-κB signal pathway. Furthermore, 12 compounds with similar scaffold with 6a were tested for anti-osteoclastogenesis. As a result, 9 compounds inhibited both NF-κB and osteoclastogenesis. Among them, compound 6b is the most potent inhibitor against NF-κB (IC50 = 2.09 μM) and osteoclast differentiation (IC50 = 0.86 μM). Further studies show that compound 6b blocks the phosphorylation of both p65 and IκBα, and suppresses NF-κB targeted gene expression without interfering MAPKs and PI3K/Akt signal transduction pathways. This study demonstrates that we can identify promising synthesized compounds with new scaffolds as therapeutic solutions against osteoclastogenesis inspired by the privileged fragment derived from natural leads.

Effects of Spirulina platensis 55% ethanol extract (SPL55) on lipid metabolism in high-fat diet-induced hyperlipidaemic rats were investigated. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry indicated that SPL55 was enriched with polyunsaturated fatty acids. Meanwhile, serum and liver lipid levels, including total triglyceride, total cholesterol, and low-density-lipoprotein cholesterol, were significantly decreased in hyperlipidaemic rats of SPL55. Analysis of tissue sections showed that SPL55 treatment could markedly inhibit hepatic lipid accumulation and steatosis. Moreover, SPL55 regulated the mRNA and protein expression levels of SREBP-1c, HMG-CoA, PEPCK, ACC, and AMPK genes involved in lipid metabolism. Furthermore, SPL55 led to decrease the abundances of Turicibacter, Clostridium_XlVa, and Romboutsia, which were positive correlation with lipid metabolism indicators, and has also enriched Alloprevotella, Prevotella, Porphyromonadaceae, and Barnesiella. These results provided evidence that SPL55 might be developed as a functional food to ameliorate lipid metabolic disorders and hyperlipidaemia.

Objective Smoking is a poor prognostic factor for lung cancer. Nicotine dependence remains the major cause of failure of smoking cessation. We investigated the risk factors for nicotine dependence in patients with lung cancer. Methods Eligible patients were identified from November 2014 to February 2015. Age, marital status, educational level, annual household income, occupation, histology of lung cancer, tumor stage, smoking status, neuron-specific enolase (NSE) level, drive gene mutations, sleep quality, and patient personality were assessed. Physical nicotine dependence was assessed by the Fagerstrom Test for Nicotine Dependence (FTND). Results In total, 202 smokers were included in this study. Univariate analysis showed that marital status and pain were significantly correlated with nicotine dependence. Pearson’s correlation analysis showed that age at the initiation of smoking, attempts to quit, NSE level, and sleep quality were significantly correlated with FTND scores. Conclusions Pain, more attempts to quit, and poorer sleep quality were significantly associated with nicotine dependence. These risk factors could help to prevent smoking in Chinese patients with lung cancer.

Effects of green macroalgae 55% ethanolic extract Enteromorpha prolifera through an ultrafiltration membrane of 3 kDa (EPE3k) on antidiabetic activity, gut microbiota, and regulation mechanism were investigated in high-fat/high-sucrose diet and streptozocin-induced diabetic mice. The structural characterizations of its major compounds in EPE3k were determined by ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry. Furthermore, the intestinal microflora modulation in diabetic mice was also investigated with high-throughput 16S rRNA gene sequencing. The proposed presence of polyphenols in EPE3k was confirmed. EPE3k could significantly decrease the fasting blood glucose and improve fasting glucose tolerance. The hypoglycemic effect of EPE3k was via activation of phosphatidylinositol 3-kinase and suppression of c-Jun N-terminal kinase in liver. EPE3k treatment significantly increased the relative abundance of Akkermansia and decreased the proportion of Alistipes and Turicibacter. The above results indicated that EPE3k could be provided as a new potential therapy for the treatment of type 2 diabetic mellitus.

Prenatal exposure to ambient fine particles (diameter < 0.25 μm, PM2.5) has been found to be associated with abnormal growth and development in offspring. However, the effects of PM2.5 on the lipid metabolism of adipose tissue in offspring are unclear. In the present study, we established a mouse model of prenatal exposure to PM2.5 by intratracheal instillation to pregnant C57BL/6 female mice with PM2.5 suspension or normal saline. We found that prenatal exposure to PM2.5 of a mouse model reduced body weight in adult male offspring after 6 weeks old. Histological analysis showed that the adipocyte size was significantly reduced in epididymal adipose tissue (eWAT) in male offspring, but not in brown adipose tissue. The expression levels of genes related to fatty acid synthesis (ACC1, ACSL1) and oxidation (PPARα) in eWAT were also significantly decreased. In addition, downregulation of pro-inflammatory cytokines (TNFα, IL-1β, IL-6) was also observed. Lipidomics analysis of eWAT demonstrated that prenatal exposure of PM2.5 reduced lysophosphatidylcholines (LPC), phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomyelins (SM), and ceramides (Cer), indicating that metabolic pathways, including SM-Cer signaling and glycerophospholipids remodeling, were disrupted. In summary, prenatal exposure to PM2.5 was associated with the dysregulations in lipid metabolism of eWAT and pro-inflammatory response in male offspring.

The novel oligosaccharides from Ulva lactuca were obtained and characterized by nuclear magnetic resonance, Fourier-transformed infrared and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. U. lactuca oligosaccharides were composed by β-D-Glcp-(1 → 4)-α-L-Rhap (2SO3−)-(1 → 2)-β-D-GlcpA-(1 → 6)-β-D-Galp-(1 → 5)-α-L-Arap-(1→ and α-L-Rhap-(1 → 2)-α-L-Rhap(3SO3−)-(1 → 6)-β-D-Glcp-(1 → 2)-β-D-Xylp-(1→ with a branch of β-D-GlcpA that connected in C4 of β-D-Glcp, respectively. After U. lactuca oligosaccharide treatment, the mean lifespan, glucose levels, and insulin resistance were improved in Caenorhabditis elegans. It has significantly enhanced the total superoxide dismutase levels and ameliorated the intracellular reactive oxygen species accumulation. U. lactuca oligosaccharides could prevent the DNA and mitochondrial damage induced by high glucose. The gene expression levels of abnormal dauer formation 16 (DAF-16), protein kinase B (AKT-1), protein skinhead-1 (SKN-1), adenosine monophosphate-activated kinase 2 (AAK-2), DAF-15, and superoxide dismutase 3 (SOD-3) were up-regulated though inhibiting the expressions of miR-48-5p, miR-67-3p, miR-124-3p, and miR-85-3p, while the gene expression levels of DAF-2, LEThal 363 (LET-363), and signal element on X 1 (SEX-1) were also up-regulated. U. lactuca oligosaccharides might be applied in the functional foods to improve glucose metabolism disorders and antioxidant systems in vivo mediated by modulation of microRNAs.

Atmospheric fine particulate matter exposure (PM2.5) can increase the incidence and mortality of heart disease, and raise the risk of fetal congenital heart defect, which have recently drawn much attention. In this study, C57BL/6 mice were exposed to PM2.5 (approximately equivalent to 174 μg/m3) by intratracheal instillation during the gestation. After birth, 10 weeks old offspring mice were divided into four groups: male exposed group (ME), female exposed group (FE), male control group (MC), female control group (FC). The pathological injury, pro-inflammatory cytokines, histone acetylation levels, and expressions of GATA-binding protein 4 (GATA4) and downstream genes were investigated. The results showed that exposure to PM2.5 in utero increased pathological damage and TNF-α and IL-6 levels in hearts of offspring mice, and effects in ME were more serious than FE. Notably, GATA4 protein levels in hearts in ME were significantly lower than that of MC, accompanied by down-regulation of histone acetyltransferase (HAT)-p300 and up-regulation of histone deacetylase-SIRT3. As GATA4 downstream genes, ratios of β-MHC gene expression to α-MHC significantly raised in ME relative to the MC. Results of chromatin immunoprecipitation (ChIP)-qPCR assay found that binding levels of acetylated histone 3 lysine 9 (H3K9ac) in GATA4 promoter region in the hearts of ME or FE were markedly decreased compared with their corresponding control groups. It suggested that maternal exposure to PM2.5 may cause cardiac injury in the offspring, heart damage of male mice was worse than female mice, in which process HAT-p300, H3K9ac, transcription factor GATA4 may play an important regulation role.

New myelin sheaths can be restored to demyelinated axons in a spontaneous regenerative process called remyelination. In general, new myelin sheaths are made by oligodendrocytes newly generated from a widespread population of adult CNS progenitors called oligodendrocyte progenitor cells (OPCs). New myelin in CNS remyelination in both experimental models and clinical diseases can also be generated by Schwann cells (SCs), the myelin-forming cells of the PNS. Fate-mapping studies have shown that SCs contributing to remyelination in the CNS are often derived from OPCs and appear not to be derived from myelinating SCs from the PNS. In this study, we address whether CNS remyelinating SCs can also be generated from PNS-derived cells other than myelinating SCs. Using a genetic fate-mapping approach, we have found that a subpopulation of nonmyelinating SCs identified by the expression of the transcription factor Foxj1 also contribute to CNS SC remyelination, as well as to remyelination in the PNS. We also find that the ependymal cells lining the central canal of the spinal cord, which also express Foxj1, do not generate cells that contribute to CNS remyelination. These findings therefore identify a previously unrecognized population of PNS glia that can participate in the regeneration of new myelin sheaths following CNS demyelination. SIGNIFICANCE STATEMENT Remyelination failure in chronic demyelinating diseases such as multiple sclerosis drives the current quest for developing means by which remyelination in CNS can be enhanced therapeutically. Critical to this endeavor is the need to understand the mechanisms of remyelination, including the nature and identity of the cells capable of generating new myelin sheath-forming cells. Here, we report a previously unrecognized subpopulation of nonmyelinating Schwann cells (SCs) in the PNS, identified by the expression of the transcription factor Foxj1, which can give rise to SCs that are capable of remyelinating both PNS and CNS axons. These cells therefore represent a new cellular target for myelin regenerative strategies for the treatment of CNS disorders characterized by persistent demyelination.

Chk1 is a cell-cycle regulator. Chk1 has been identified in organisms ranging from yeast to humans, but few researchers have studied Chk1 in shrimps. We cloned Chk1 from the black tiger shrimp (Penaeus monodon). The full-length cDNA sequence of PmChk1 had 3,334 base pairs (bp), with an open reading frame of 1,455 bp. The complete genomic sequence of PmChk1 (11,081 bp) contained 10 exons separated by nine introns. qRT-PCR showed that PmChk1 was highly expressed in the ovaries and gills of P. monodon. The lowest PmChk1 expression was noted in stage III of ovarian development in P. monodon. PmChk1 expression decreased significantly after injection of 5-hydroxytryptamine and eyestalk ablation in P. monodon ovaries. RNA interference experiments were undertaken to examine the expression of PmChk1, PmCDC2, and PmCyclin B. PmChk1 knockdown in the ovaries and hepatopancreas by dsRNA-Chk1 was successful. The localization and level of PmChk1 expression in the hepatopancreas was studied using in situ hybridization, which showed that data were in accordance with those of qRT-PCR. The Gonadosomatic Index of P. monodon after dsRNA-Chk1 injection was significantly higher than that after injection of dsRNA-GFP or phosphate-buffered saline. These data suggest that PmChk1 may have important roles in the ovarian maturation of P. monodon.

As a unique atypical 2-Cys Peroxiredoxin (Prx) of the Prx-like superfamily, Peroxiredoxin5 (Prx5) possesses special properties, such as its enzymatic mechanism, wide subcellular distribution and high affinity for peroxides and peroxynitrite. Prx5 plays a crucial role in oxidative stress, immune responses, cell apoptosis, proliferation, differentiation, intracellular signaling, the modulation of gene expression, ecdysis, etc. In this paper, we obtained a full-length Prx5 cDNA sequence (designated PmPrx5) from black tiger shrimp (P. monodon). The full-length PmPrx5 cDNA sequence was 1686 bp containing a 5' untranslated region (UTR) of 76 bp with two nucleotide sequences (AAA), a 3' UTR of 1040 bp with a poly (A) tail and two canonical polyadenylation signal sequences (AATAAA), and an open reading frame of 570 bp encoding 189 amino acid residues with a predicted molecular mass of 20 kDa and a theoretical isoelectric point of 6.29. Phylogenetic trees and multiple sequence alignment showed that the PmPrx5 had strong homology with Prx5 proteins from other species, such as similarity with Palaemon carinicauda (69%) and Macrobrachium rosenbergii (69%), containing the highly conserved functional domain. PmPrx5 mRNA was ubiquitously detected in all tested tissues. After P. monodon was exposed to pathogenic bacteria, osmotic pressure, acidity and alkalinity and the heavy metal, the mRNA expression of PmPrx5 in the gills and hepatopancreas was significantly enhanced (P 0.01) because of the immune response and declined with heavy metal copper and cadmium challenges as time progressed. The recombinant PmPrx5 protein purified in E. coli (DE3) was further confirmed to exhibit antioxidant activity and antibacterial properties to a certain extent using a bacterial growth inhibition test in both liquid and solid cultures in vitro. E. coli transformed with pRSET-PmPrx5 were dramatically protected in response to metal toxicity stress. Thus, PmPrx5 may be developed as a potential therapeutic drug against pathogenic bacteria and as a biomarker for pollutant levels. This work offers useful clues to further explore the functional mechanism of Prx5 in marine shrimp immunity.

The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.

Cystathionine-γ-lyase (CSE) is a major endogenous enzyme producing H2S which, as a third gasotransmitter, plays important roles in many physiological and pathological processes. The mechanism of regulating CSE gene expression is unclear and the roles of CSE/H2S in tumor also have not got a profound understanding, especially in colon cancer. Our study demonstrated that CSE gene expression was regulated by the Wnt pathway on transcriptional level. Activating the Wnt pathway by either Wnt3a or LiCl increased CSE mRNA and protein levels, while siRNA-mediated silence of β-catenin decreased CSE mRNA and protein levels. XAV939 treatment which accelerated β-catenin degradation could reduce CSE protein level. To reveal the mechanism, two TCF/LEF binding sites were found in CSE promoter whose activity had a positive response to β-catenin overexpression in 293T cells. Mutations of TCF/LEF binding sites led to an increase of the promoter activity. It indicated that TCF/LEF likely acted as a repressor to CSE gene transcription, and Wnt signal contributed to free β-catenin accumulation to possibly relieve the repression. Either knockdown of CSE by shRNA (shCSE) or its inhibition by PAG decreased SW480 cell proliferation, migration, and tumor xenograft growth in nude mice. In conclusion, we have demonstrated that the Wnt pathway regulates CSE gene expression on transcriptional level and CSE/H2S plays important roles in colon cancer.

Scope In this study, the antidiabetic activity of Lessonia nigrescens ethanolic extract (LNE) is investigated in streptozotocin (SZT)-induced type 2 diabetic mice fed with a high-sucrose/high-fat diet. Methods and results Ultra high performance liquid chromatography coupled with photo-DAD and electospray ionization-mass spectrometry (ESI-MS) is employed to analyze the major compounds in LNE. The components of the intestinal microflora in type 2 diabetic mice are analyzed by high-throughput next-generation 16S rRNA gene sequencing. Fasting blood glucose levels in diabetic mice are significantly decreased after LNE administration. The histology reveals that LNE could protect the cellular architecture of liver and kidney. LNE treatment significantly increases Bacteroidetes and decreases Firmicutes populations in intestinal microflora. Specifically, It could selectively enrich the amounts of beneficial bacteria, Barnesiella, as well as reduce the abundances of Clostridium and Alistipes. The increased gene and protein expression levels of phosphatidylinositol 3-kinase (PI3K) in the liver are observed in LNE treatment groups, while the expressions of c-Jun N-terminal kinase (JNK) are significantly downregulated. Conclusion The above findings suggest that LNE could be considered as a functional food for reducing blood glucose and regulating intestinal microflora.

Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)-E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA-E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA-GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon.

The study investigates the expression and clinical role of GLP-1R in intrahepatic cholangiocarcinoma (ICC) tissues. ICC tissue, tissue around tumour and normal liver tissue samples from 176 ICC patients were investigated for GLP-1R expression by immunohistochemistry and western blots. Expression levels were correlated to clinical variables and to the postoperative outcome. High GLP-1R expression levels were detected in tumor tissue samples. Kaplan-Meier method was used for survival analysis of patients follow-up data. Results showed that median survival time of patients with high GLP-1R positive expression in ICC tissue were 22 months. Median survival time of patients with low GLP-1R positive expression in ICC tissue were 19.8 months. There wasn't statistical difference (p = 0.332) between two groups. Immunohistochemistry semi-quantitative analysis showed that tissue differentiation is not prognostic risk factors. In patients with GLP-1R positive expression in ICC tissue, lymph node metastasis was important prognostic factors (p = 0.001). Although statistical analysis showed that GLP-1R can not be judged as a risk prognostic factors, GLP-1 might become a new target for therapy of ICC.

The guided migration of neural cells is essential for repair in the central nervous system (CNS). Oligodendrocyte progenitor cells (OPCs) will normally migrate towards an injury site to re-sheath demyelinated axons; however the mechanisms underlying this process are not well understood. Endogenous electric fields (EFs) are known to influence cell migration in vivo, and have been utilised in this study to direct the migration of OPCs isolated from neonatal Sprague-Dawley rats. The OPCs were exposed to physiological levels of electrical stimulation, and displayed a marked electrotactic response that was dependent on β1 integrin, one of the key subunits of integrin receptors. We also observed that F-actin, an important component of the cytoskeleton, was re-distributed towards the leading edge of the migrating cells, and that this asymmetric rearrangement was associated with β1 integrin function.

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Ultra-high performance liquid chromatography coupled with photo-diode array detector and electrospray ionization-mass spectrometry was employed to analyze the major fatty acids in Spirulina platensis 95% ethanol extract (SPL95). The effects of SPL95 on hepatoprotection were evaluated, including liver tissue histopathology, liver, and serum biochemical analysis. The active principle of SPL95 revealed a hypolipidemic effect, as indicated by down-regulating the mRNA and protein levels of sterol regulatory element-binding transcription factor-1c, 3-hydroxy-3-methyl glutaryl coenzyme A reductase, acetyl CoA carboxylase pathway, and upregulating adenosine 5&prime, monophosphate-activated protein kinase-&alpha, in liver. SPL95 enriched the beneficial bacteria, including Prevotella, Alloprevotella, Porphyromonadaceae, Barnesiella, and Paraprevotella. Treatment with SPL95 led to a decrease in microbes, such as Turicibacter, Romboutsia, Phascolarctobacterium, Olsenella, and Clostridium XVIII, which were positively correlated with serum triglyceride, total cholesterol, and low-density-lipoprotein cholesterol levels, but negatively correlated with the serum high-density-lipoprotein cholesterol levels. These results provide evidence that the fatty acid from SPL95 may be used as a novel adjuvant therapy and functional food to regulate gut microbiota in obese and diabetic individuals.

Adaptor proteins are involved in the assembly of various intracellular complexes and the regulation of cellular functions. Membrane-associated guanylate kinase inverted 2 (MAGI2), also known as synaptic scaffolding molecule (S-SCAM), plays a critical role in signal transduction by assembling and anchoring its ligands. However, the role of MAGI2 in mediating apoptosis remains largely unknown. In the present study, BEL-7404 human hepatocellular carcinoma cells were transfected with a plasmid containing myc-MAGI2 or an empty plasmid and cell viability was then determined using the Cell Counting kit-8. Apoptosis was also detected using an Annexin V apoptosis assay. The cells were then treated with various doses of staurosporine (STS) for different periods of time. The overexpression of myc-MAGI2 was found to sensitize the BEL-7404 cells to apoptosis in response to STS in a time- and dose-dependent manner. Our results demonstrated that MAGI2 enhanced STS-induced apoptosis by increasing the protein expression of cytoplasmic phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and decreasing its protein degradation. The apoptotic sensitivity of the cells caused by the overexpression of myc-MAGI2 was reversed by the silencing of PTEN expression by PTEN siRNA, thus revealing a momentous role of PTEN in the enhancement of the sensitivity of cancer cells to STS-induced apoptosis by MAGI2. Finally, we observed that the MAGI-PTEN complex triggered by MAGI2 overexpression reduced the phosphorylation levels of AKT. These results suggest that MAGI2 overexpression enhances the sensitivity of cancer cells harboring ectopic PTEN to STS-induced apoptosis.

Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.

Several androgen receptor (AR) antagonists are clinically prescribed to treat prostate cancer. Unfortunately, many patients become resistant to the existing AR antagonists. To overcome this, a novel AR antagonist candidate called DIMN was discovered by our research group in 2013. In order to develop compounds with improved potency, we designed novel DIMN derivatives based on a docking study and substituted carbons with heteroatom moieties. Encouraging in vitro results for compounds 1b, 1c, 1e, 3c, and 4c proved that the new design was successful. Among the newly synthesized compounds, 1e exhibited the strongest inhibitory effect on LNCaP cell growth (IC50=0.35μM) and also acted as a competitive AR antagonist with selectivity over the estrogen receptor (ER) and the glucocorticoid receptor (GR). A docking study of compound 1e fully supported these biological results. Compound 1e is considered to be a novel, potent and AR-specific antagonist for treating prostate cancer. Thus, our study successfully applied molecular modeling and bioisosteric replacement for hit optimization. The methods here provide a guide for future development of drug candidates through structure-based drug discovery and chemical modifications.

The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed d -glucosyl-β-(1–4)- d -glucose to glucose. Using p-nitrophenyl-β- d -glucoside as substrate, Km was 0.54 and 2.11 mM, and kcat/Km was 1489 and 787 mM−1 min−1 for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30 °C, while the optimum values for Bgl1E were pH 10.0 and 25 °C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl3 and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5–10.5), making it a potential candidate for industrial applications.

X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. High-efficiency transduction of ALD CD34(+) peripheral blood mobilized cells was achieved using an HIV-based vector driving ALD gene expression under the elongation factor 1 alpha promoter and a protocol without prestimulation of CD34(+) cells with cytokines prior to transduction to preserve their stem cell properties. Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.

Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels ∼4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c increased ACCI mRNA levels. Finally, glucose also stimulated SREBP1c transcription, although this effect was independent of insulin release. These data suggest that glucose regulates ACCI gene expression in the β-cell by complex mechanisms that may involve the covalent modification of SREBP1c. However, overexpression of SREBP1c also decreased glucose-stimulated insulin release, implicating SREBP1c induction in β-cell lipotoxicity in some forms of type 2 diabetes.

Smoking is the primary cause of lung cancer and is linked to 85% of lung cancer cases. However, how lung cancer develops in patients with smoking history remains unclear. Systems approaches that combine human protein-protein interaction (PPI) networks and gene expression data are superior to traditional methods. We performed these systems to determine the role that smoking plays in lung cancer development and used the support vector machine (SVM) model to predict PPIs. By defining expression variance (EV), we found 520 dynamic proteins (EV>0.4) using data from the Human Protein Reference Database and Gene Expression Omnibus Database, and built 7 dynamic PPI subnetworks of lung cancer in patients with smoking history. We also determined the primary functions of each subnetwork: signal transduction, apoptosis, and cell migration and adhesion for subnetwork A; cell-sustained angiogenesis for subnetwork B; apoptosis for subnetwork C; and, finally, signal transduction and cell replication and proliferation for subnetworks D–G. The probability distribution of the degree of dynamic protein and static protein differed, clearly showing that the dynamic proteins were not the core proteins which widely connected with their neighbor proteins. There were high correlations among the dynamic proteins, suggesting that the dynamic proteins tend to form specific dynamic modules. We also found that the dynamic proteins were only correlated with the expression of selected proteins but not all neighbor proteins when cancer occurred.

To study the gene expression profiles in dendritic cells (DCs) from a hepatitis B surface antigen (HBsAg) positive host, transcriptional analysis of bone marrow -derived DCs from a lineage of HBsAg transgenic mice (#59) was compared to DCs from normal mice. Among the immune-related genes, 12 were up-regulated, and 14 were down-regulated in transgenic mice relative to those of normal mice. The up-regulated genes include genes encoding immunoglobulin, histocompatibility 2 (K region), and several complement component genes, while the down-regulated genes include the TAP1 (transporters associated with antigen processing gene-1), interferon induced gene (Ifi203), chemokine (C-X-C) ligands and leukocyte-immunoglobulin-like genes, Lck-interacting transmembrane adaptor genes and histocompatibility 2 (Q region and T region). Since an immunogenic complex containing HBsAg-anti-HBs has been used as a therapeutic vaccine for clinical trial in chronic hepatitis B patients, DCs from #59 were incubated with immunogenic complex compared to those incubated with HBsAg alone. The immune-related six genes up-regulated with immunogenic complex treatment were Fcgr2b, Cxcl2, Fth1, Clec4n, Lilrb4, and Dbh, with Fcgr2b (Fc gamma receptor IIB) being the highest up-regulated gene. Interestingly, levels of Fcgr2b were found up-regulated in patients with chronic hepatitis B undergoing immunogenic complex immunization, which returned to baseline when immunization was discontinued. In conclusion, by transcriptional analysis, immunogenic complex induced up-regulation of Fcgr2b expression both in dendritic cells from an HBsAg transgenic mouse model and peripheral B cells from patients with chronic hepatitis B, which indicates that Fcgr2b is one of the key molecules up-regulated by immunogenic complex.

To construct an eukaryotic recombinant expression vector for retinoblastoma 1 gene (RB-1) and investigate the role of RB-1 in prostate cancer.The coding sequence of RB-1 gene tagged with FLAG was amplified from the plasmid CMV-RB by PCR method. The fragment was cloned into CMV expression vector and identified by restriction enzyme digestion and sequence analysis. Western Blotting was used to detect RB-1 expression and immunofluorescence was used to observe RB-1 distribution in PC-3 cells transfected with the recombinant.The expression vector CMV-FLAG-RB was successfully constructed as confirmed by PCR, endonuclease digestion and DNA sequence analysis. RB-1 protein was highly expressed and showed a nuclear distribution in PC-3 cells transfected with the recombinant.The eukaryotic expression vector for RB-1 has been successfully constructed and can be efficiently expressed in PC-3 cells. The expression of RB-1 is located in the cell nuclei.

Purpose Dominant Stargardt macular dystrophy (STGD3) is caused by several different mutations in a gene named ELOVL4, which shares sequence homologies with a family of genes that encode proteins involved in the ELOngation of Very Long chain fatty acids. Studies have suggested that patients with STGD3 have aberrant metabolism of docosahexaenoic acid (DHA, 22:6n3), the major polyunsaturated fatty acid (PUFA) in retinal rod outer segment membranes. We tested the effect of DHA on the progression of retinal degeneration in transgenic mice that express one of the mutations identified in STGD3. Methods Transgenic mice expressing mutant human ELOVL4 (TG2) were bred to mice expressing the fat-1 protein, which can convert n6 to n3 PUFA. Mice were maintained on an n3-deficient diet containing 10% safflower oil (SFO, enriched in n6 PUFA; n6/n3=273) so that four experimental groups were produced that differed only in levels of n3 PUFA and expression of the hELOVL4 transgene. These groups were identified by genotyping and named Fat1+/TG2+, Fat1–/TG2+, Fat1+/TG2–, and Fat1–/TG2–. All were continued on the SFO diet for 4 to 16 weeks such that those not expressing Fat1 would be deficient in n3 fatty acids. At both time points, animals were analyzed for retinal function by electroretinography (ERG), photoreceptor cell viability by outer nuclear layer (ONL) thickness measurements, fatty acid profiles in several tissues, and rhodopsin levels. Results Mice expressing the fat-1 transgene had significantly higher levels of n3 PUFA, primarily DHA, in retina, liver, and plasma lipids at 4 and 16 weeks of age. Retinal DHA levels in fat-1 mice were twice those of controls. By 16 weeks of age, mice expressing the mutant hELOVL4 transgene had a significantly greater loss of photoreceptor cells, reduced ERG amplitudes, and lower rhodopsin levels than control mice. There was no effect of retinal fatty acids on the rate of degeneration of retinas expressing the ELOVL4 transgene. Conclusions We found no evidence that high levels of DHA in retinal membranes protected photoreceptor cells expressing mutant ELOVL4 from retinal degeneration. We conclude that DHA is not beneficial for the treatment of retinal degeneration in this animal model of human STGD3 macular dystrophy.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) affect proliferation and survival of many cell types, but their role in the maintenance of olfactory mucosa cells remains unclear. In the neonatal mouse olfactory mucosa, cell proliferation mainly occurs in the neuroepithelium and, to a lesser extent, in the lamina propria. To establish whether bFGF and EGF affect proliferation and/or survival of these cells, we isolated olfactory mucosa cells from the neonatal mouse and cultured them as free-floating spheres under bFGF or EGF stimulation. Our data demonstrate that bFGF is a mitogen for the rapidly dividing cells (olfactory neuronal precursors and olfactory ensheathing cells), and also a survival factor for both slowly and rapidly dividing cells of the olfactory mucosa. In contrast, EGF appears to be primarily a survival factor for both the olfactory stem and precursor cells.

The genome of hepatitis B virus (HBV) consists of four open reading frames, encoding the envelope proteins (Pre-S/S), the core proteins (Pre-C/C), the polymerase (P) and the transactivating X protein (X). In the sera of HBV-infected patients, hepatitis B surface antigen (HBsAg) particles without the viral genome can outnumber virions by more than 1000-fold. To analyse the interactions between HBsAg and host cells, global gene-expression profiles of a small HBsAg (SHBs)-secreting stable cell line (HepG2-S-G2) and its counterpart control cell line (HepG2-Neo-F4) were compared. Marked upregulation of lymphoid enhancer-binding factor 1 (LEF-1), a transcription factor in the Wnt pathway, was found in SHBs-expressing cells and was confirmed by interference experiments with small interfering RNA. However, compared with the control cells, HepG2-S-G2 did not show higher proliferative competence in culture or increased tumorigenesis in nude mice. A possible mechanism to explain the discrepancy between the upregulation of LEF-1 and the lack of increased tumorigenesis is SHBs expression resulting in altered expression and distribution of LEF-1 protein in cell compartments and upregulation of LEF-1 isoforms that could suppress, rather than enhance, the Wnt pathway.

To achieve high expression of human renal cell carcinoma-associated antigen G250 in Escherichia coli.The gene fragments encoding the protein obtained by PCR was cloned into prokaryotic expression vector pET32a(+) and expressed in E. coli Rosseta. The immunogenicity of the recombinant protein was evaluated by Western blotting.The plasmid pET32a(+)/G250 was constructed and expressed in E. coli Rosseta successfully. Western blot analysis showed that the recombinant protein could be specifically recognized by monoclonal antibody M75.Efficient G250 expression is achieved in prokaryotic expression system, which may facilitate further functional study of the protein and its monoclonal antibody preparation.

CNS remyelination occurs more rapidly in young adult rats than in old rats. Since the inflammatory response initiated by demyelination is an important trigger for remyelination, we address whether ageing changes in remyelination are associated with changes in the inflammatory response. Using a toxin model of demyelination, where the inflammatory response largely comprises macrophages, we show that there is a delay in both recruitment and activation of OX-42+ and macrophage scavenger receptor B+ macrophages following demyelination in older rats (10–13 months) compared to young rats (8–10 weeks). This difference is associated with a slower onset of increased expression of several chemokine mRNAs. However, many inflammatory cytokines have similar mRNA expression patterns, with the exception of IL-1β, IL-6 and TNF-α, which have prolonged expression in the older animals. Differences in IL-1β mRNA expression, a cytokine specifically implicated in CNS remyelination, are not reflected in differences in protein expression detected by immunocytochemistry. These data relate the age-associated delay in remyelination efficiency to changes in the macrophage and inflammatory mediator response to demyelination.

Colonic epithelium is known to secrete both Cl(-) and HCO(3)(-), but the secretory mechanisms of different colonic cell types are not fully understood. The present study aimed to investigate the differential activation of Cl(-) and HCO(3)(-) secretion by tetramethylpyrazine (TMP) in human crypt-like cell line, T84, and villus-like cell line, Caco-2, in comparison to the TMP-induced secretory response in freshly isolated rat colonic mucosa.Colonic epithelial anion secretion was studied by using the short circuit current (I(SC)) technique. RT-PCR was used to examine the expression of Na(+)-HCO(3)(-)-cotranspoter in different epithelial cell types.TMP produced a concentration-dependent I(SC) which was increase in both T84 and Caco-2 cells. When extracellular Cl(-) was removed, TMP-induced I(SC) was abolished by 76.6% in T84 cells, but not in Caco-2 cells. However, after both Cl(-) and HCO(3)(-) were removed, TMP-induced I(SC) in Caco-2 cells was reduced to 10%. Bumetanide, an inhibitor of Na(+)-K(+)-Cl(-)-cotranspoter, inhibited the TMP-induced I(SC) by 96.7% in T84 cells, but only 47.9% in Caco-2 cells. In the presence of bumetanide and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid, an inhibitor of Na(+)-HCO(3)(-) cotransporter, inhibited the TMP-induced current in Caco-2 cells by 93.3%. In freshly isolated rat colonic mucosa, TMP stimulated distinct I(SC) responses similar to that observed in T84 and Caco-2 cells depending on the concentration used. RT-PCR revealed that the expression of Na(+)-HCO(3)(-) cotransporter in Caco-2 cells was 4-fold more greater than that in T84 cells.TMP exerts concentration-dependent differential effects on different colonic cell types with stimulation of predominant Cl(-) secretion by crypt cells at a lower concentration, but predominant HCO(3)(-) secretion by villus cells at a higher concentration, suggesting different roles of these cells in colonic Cl(-) and HCO(3)(-) secretion.