Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

Construction of miRNA-mRNA network for the identification of key biological markers and their associated pathways in IgA nephropathy by employing the integrated bioinformatics analysis

BackgroundAbout half-century ago, Immunoglobulin A nephropathy (IgAN) was discovered as a complicated disease with frequent clinical symptoms. Until now, exact mechanism underlying the pathogenesis of IgAN is poorly known. Therefore, current study was aimed to understand the molecular mechanism of IgAN by identifying the key miRNAs and their targeted hub genes. The key miRNAs might contribute to the diagnosis and therapy of IgAN, and could turn out to be a new star in the field of IgAN.MethodsThe microarray datasets were downloaded from Gene Expresssion Omnibus (GEO) database and analyzed using R package (LIMMA) in order to obtain differential expressed genes (DEGs). Then, the hub genes were identified using cytoHubba plugin of cytoscpae tool and other bioinformatics approaches including protein-protein interaction (PPI) network analysis, module analysis, and miRNA-hub gene network construction was also performed.ResultsA total of 348 DEGs were identified, of which 107 were upregulated genes and 241 were downregulated genes. Subsequently, the 12 overlapped genes were predicted from cytoHubba, and considered as hub genes. Moreover, a network among miRNA-hub genes was created to explore the correlation between the hub genes and their targeted miRNAs. Network construction ultimately lead to the identification of nine gene named FN1, EGR1, FOS, JUN, SERPINE1, MMP2, ATF3, MYC, and IL1B and one novel key miRNA namely, has-miR-144-3p as biomarker for diagnosis and therapy of IgAN.ConclusionThis study updates the information and yield a new perspective in context of understanding the pathogenesis and development of IgAN. In future, key miRNAs might be capable of improving the personalized detection and therapies for IgAN. In vivo and in vitro investigation of miRNAs and pathway interaction is essential to delineate the specific roles of the novel miRNAs, which may help to further reveal the mechanisms underlying IgAN.     

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73 U/mg) at pH5 and 50 °C. The K_m and V_max with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20 mM and 2.41 U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140 mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

Characterization of Acinetobacter chengduensis sp. nov., isolated from hospital sewage and capable of acquisition of carbapenem resistance genes

Two strains of the genus Acinetobacter, WCHAc060005^T and WCHAc060007, were isolated from hospital sewage in China. The two strains showed different patterns of resistance to clinically important antibiotics and their taxonomic positions were investigated. Cells are Gram-negative, obligate aerobic, non-motile, catalase-positive and oxidase-negative coccobacilli. A preliminary analysis based on the 16S rRNA gene sequences indicated that the two strains had the highest similarity to Acinetobacter cumulans WCHAc060092^T (99.02%). Whole-genome sequencing of the two strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and in silico DNA–DNA hybridization values between the two new genomes were 99.77% and 98.7% respectively, whereas those between the two genomes and the known Acinetobacter species were <88.93% and <34.0%, respectively. A total of 7 different genes were found in the two genome sequences which encode resistance to five classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, and quinolones. In addition, the combination of their ability to assimilate gentisate, but not l-glutamate and d,l-lactate could distinguish the two strains from all known Acinetobacter species. Based on these combined data, we concluded that the two strains represent a novel species of the genus Acinetobacter, for which the name Acinetobacter chengduensis sp. nov. is proposed. The type strain is WCHAc060005^T (CCTCC AB 2019139 = GDMCC 1.1622 = JCM 33509).     

Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

MicroRNAs (miRNAs) are small (～22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.     

MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas

MicroRNAs (miRNA) are small non-coding RNAs that inhibit gene expression through binding to complementary messenger RNA sequences. miRNAs have been predicted to target genes important for pancreas development, proper endocrine cell function and metabolism. We previously described that miRNA-7 (miR-7) was the most abundant and differentially expressed islet miRNA, with 200-fold higher expression in mature human islets than in acinar tissue. Here we have analyzed the temporal and spatial expression of miR-7 in human fetal pancreas from 8 to 22 weeks of gestational age (wga). Human fetal (8–22 wga) and adult pancreases were processed for immunohistochemistry, in situ hybridization, and quantitative RT-PCR of miRNA and mRNA. miR-7 was expressed in the human developing pancreas from around 9 wga and reached its maximum expression levels between 14 and 18 wga, coinciding with the exponential increase of the pancreatic endocrine hormones. Throughout development miR-7 expression was preferentially localized to endocrine cells and its expression persisted in the adult pancreas. The present study provides a detailed analysis of the spatiotemporal expression of miR-7 in developing human pancreas. The specific localization of miR-7 expression to fetal and adult endocrine cells indicates a potential role for miR-7 in endocrine cell differentiation and/or function. Future functional studies of a potential role for miR-7 function in islet cell differentiation and physiology are likely to identify novel targets for the treatment of diabetes and will lead to the development of improved protocols for generating insulin-producing cells for cell replacement therapy.     

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.     

Genetic variation in a miR-335 binding site in BIRC5 alters susceptibility to lung cancer in Chinese Han populations

Polymorphisms in 3′ untranslated region (UTR) of cancer-related genes might affect their regulation by microRNAs (miRNAs) and thereby contribute to carcinogenesis. In this study, we screened single nucleotide polymorphisms (SNPs) in 3′ UTR of cancer-related genes and investigated their effects on risk of lung cancer. First, we genotyped seven SNPs in a Chinese Han population with 600 lung cancer patients and 600 matched healthy controls and found that compared with the TT genotype of rs2239680 in 3′ UTR of baculoviral IAP repeat containing 5 (BIRC5), C allele was associated with a significantly increased risk of lung cancer and advanced pathologic stage, with the odds ratio for participants carrying the CT or CC genotype being 1.50 [95% confidence interval (CI) 1.20–1.89, P < 0.01] and 2.29 (95% CI 1.64–3.18, P < 0.01), respectively. These results were further replicated and confirmed in another independent population with 1000 lung cancer cases and 1000 matched healthy controls. In support of the postulation that the 3′ UTR SNP may directly affect miRNA-binding site, reporter gene assays indicated BIRC5 was a direct target of miR-335, and the rs2239680 T > C change resulted in altered regulation of BIRC5 expression. Moreover, BIRC5 was over expressed in lung cancer tissues compared with the normal lung tissues, and the protein levels of BIRC5 correlated with SNP genotypes in normal lung tissues. Our findings defined a 3′ UTR SNP in human BIRC5 oncogene that may increase individual susceptibility to lung cancer probably by attenuating the interaction between miR-335 and BIRC5.     

Molecular characterization of aromatase inhibitor-resistant,tamoxifen-resistant and LTEDaro cell lines

To determine potential genes involved in mediating resistance to aromatase inhibitors (AIs), a microarray study was performed using MCF-7aro (aromatase overexpressing) cells that are resistant to letrozole (T + LET R), anastrozole (T + ANA R) and exemestane (T + EXE R), as well as LTEDaro and tamoxifen-resistant (T + TAM R) lines for comparison. Based on hierarchical clustering, estrogen-responsive genes were found to be differentially expressed in AI-resistant lines versus LTEDaro and T + TAM R. Additional genome-wide analysis showed that gene expression profiles of the non-steroidal AI-resistant lines were most closely correlated and that T + EXE R lines exhibit differing profiles. Also, LTEDaro and T + TAM R lines are inherently different from expression profiles of AI-resistant lines. Further characterization of these resistant lines revealed that T + LET R, T + ANA R and LTEDaro cells contain a constitutively active estrogen receptor α (ERα) that does not require the ligand estrogen for activation. Ligand-independent activation of ERα does not activate identical estrogen-responsive gene profiles in AI-resistant lines as in LTEDaro lines, thereby establishing differing mechanisms of resistance. This ligand-independent activation of ER was not observed in the parental cell lines MCF-7aro, T + EXE R or T + TAM R cells. Based on the steroidal structure of EXE, our laboratory has shown that this AI has weak estrogen-like properties, and that EXE resistance involves an ER-dependent crosstalk with EGFR growth factor signaling. Recent studies in our laboratory pertaining to pre-clinical models of AI treatment revealed that intermittent use of EXE delays the onset of acquired resistance in comparison to continuous treatment. Specific molecular mechanisms involved in intermittent use of EXE are currently being explored, based on microarray gene expression profiling. Lastly, our laboratory has initiated a study of microRNAs and their potential role in regulating target genes involved in AI-resistance. Overall, we propose a model of acquired resistance that progresses from hormone-dependence (T + TAM R and T + EXE R) to hormone-independence (T + LET R and T + ANA R), eventually resulting in hormone-independence that does not rely on conventional ER signaling (LTEDaro).     

Acinetobacter cumulans sp. nov., isolated from hospital sewage and capable of acquisition of multiple antibiotic resistance genes

We studied the taxonomic position of six phenetically related strains of the genus Acinetobacter, which were recovered from hospital sewage in China and showed different patterns of resistance to clinically important antibiotics. Whole-genome sequencing of these strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and digital DNA–DNA hybridization values between the six new genomes were 97.25–98.67% and 79.2–89.3%, respectively, whereas those between them and the genomes of the known species were ≤78.57% and ≤28.5%, respectively. The distinctness of the strains at the species level was also supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by MALDI-TOF MS. Moreover, the strains displayed a catabolically unique profile and could be differentiated from the phylogenetically closest species at least by their inability to grow on d,l-lactate. A total of 18 different genes were found in the six genome sequences which encode resistance to seven classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, or aminoglycosides. These genes occurred in five different combinations, with three to 10 different genes per strain. We conclude that the six strains represent a novel Acinetobacter species, for which we propose the name Acinetobacter cumulans sp. nov. to reflect its ability to acquire and cumulate diverse resistance determinants. The type strain is WCHAc060092^T (ANC 5797^T = CCTCC AB 2018119^T = GDMCC 1.1380^T = KCTC 62576^T).     

Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice

Zuotai (mainly β-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl₂) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl₂ and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (β-HgS, 30 mg/kg), α-HgS (HgS, 30 mg/kg), HgCl₂ (33.6 mg/kg), or MeHg (3.1 mg/kg) for 7 days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1–4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl₂. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl₂ and MeHg, but not by Zuotai and HgS. HgCl₂ decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl₂ and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl₂ and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.