Search

High-throughput transcriptomics has revolutionised the field of transcriptome research by offering a cost-effective and powerful screening tool. Standard bulk RNA sequencing (RNA-Seq) enables characterisation of the average expression profiles for individual samples and facilitates identification of the molecular functions associated with genes differentially expressed across conditions. RNA-Seq can also be applied to disentangle splicing variants and discover novel transcripts, thus contributing to a comprehensive understanding of the transcriptome landscape. A closely related technique, single-cell RNA-Seq, has enabled the study of cell-type-specific gene expressions in hundreds to thousands of cells, aiding the exploration of cell heterogeneity. Nowadays, bulk RNA-Seq and single-cell RNA-Seq serve as complementary tools to advance and accelerate the development of transcriptome-based resources. This Collection illustrates how the current global research community makes use of these techniques to address a broad range of questions in life sciences. It demonstrates the usefulness and popularity of high-throughput transcriptomics and presents the best practices and potential issues for the benefit of future end-users. [ABSTRACT FROM AUTHOR]

The common management practices of dairy calves leads to increased starch concentration in feed, which subsequently may cause rumen acidosis while on milk and during weaning. Until recently, few attempts were undertaken to understand the health risks of prolonged ruminal acidosis in post weaning calves. Resultantly, the molecular changes in the digestive tracts in post-weaning calves with ruminal acidosis remain largely unexplored. In this study, we investigated the liver transcriptome changes along with its correlation with the rumen microbial rRNA expression changes in young calves using our model of feed induced ruminal acidosis. In this model, new born calves were fed a highly processed, starch-rich diet starting from one week of age through 16 weeks. A total of eight calves were involved in this study. Four of them were fed the acidosis-inducing diet (Treated) and the rest of the four were fed a standard starter diet (Control). Liver and rumen epithelial tissues were collected at necropsy at 17 weeks of age. Transcriptome analyses were carried out in the liver tissues and rRNA meta-transcriptome analysis were done using the rumen epithelial tissues. The correlation analysis was performed by comparing the liver mRNA expression with the rumen epithelial rRNA abundance at genus level. Calves with induced ruminal acidosis had significantly lower ruminal pH in comparison to the control group, in addition to significantly less weight-gain over the course of the experiment. In liver tissues, a total of 428 differentially expressed genes (DEGs) (fold-change, FC ≥ 1.5; adjusted P ≤ 0.1) were identified in treated group in comparison to control. Biological pathways enriched by these DEGs included cellular component organization, indicating the impact of ruminal acidosis on liver development in young calves. Specifically, the up-regulated genes were enriched in acute phase response (P < 0.01), pyruvate metabolic process (P < 0.01) and proton-acceptors (P ≪ 0.001), indicating the liver's response to feed induced acidosis at the transcriptome level. Twelve transferase activity related genes had significant correlation with rumen microbial rRNA expression changes. Among these genes, two up-regulated genes were reported with involvement in lipid metabolism in the liver, implying the direct effect of feed-induced acidosis on both the rumen microbial community and liver metabolism. Our study provides insight into the physiological remodeling in the liver resultant from the prolonged acidosis in post weaning calves, which may facilitate future RNA-seq based diagnosis and precision management of rumen acidosis in dairy calves. [ABSTRACT FROM AUTHOR]

This study sought to characterize differences in gene content, regulation and structure between taurine cattle and river buffalo (one subspecies of domestic water buffalo) using the extensively annotated UMD3.1 cattle reference genome as a basis for comparisons. We identified 127 deletion CNV regions in river buffalo representing 5 annotated cattle genes. We also characterized 583 merged mobile element insertion (MEI) events within the upstream regions of annotated cattle genes. Transcriptome analysis in various tissue types on river buffalo confirmed the absence of four cattle genes. Four genes which may be related to phenotypic differences in meat quality and color, had upstream MEI predictions and were found to have significantly elevated expression in river buffalo compared with cattle. Our comparative alignment approach and gene expression analyses suggested a functional role for many genomic structural variations, which may contribute to the unique phenotypes of river buffalo. [ABSTRACT FROM AUTHOR]

Copy number variation (CNV), generated through duplication or deletion events that affect one or more loci, is widespread in the human genomes and is often associated with functional consequences that may include changes in gene expression levels or fusion of genes. Genome-wide association studies indicate that some disease phenotypes and physiological pathways might be impacted by CNV in a small number of characterized genomic regions. However, the pervasiveness and full impact of such variation remains unclear. Suitable analytic methods are needed to thoroughly mine human genomes for genomic structural variation, and to explore the interplay between observed CNV and disease phenotypes, but many medical researchers are unfamiliar with the features and nuances of recently developed technologies for detecting CNV. In this article, we evaluate a suite of commonly used and recently developed approaches to uncovering genome-wide CNVs and discuss the relative merits of each. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies revealed that two genes-sanU and sanV were associated with nikkomycin biosynthesis in Streptomyces ansochromogenes. A plasmid used to increase an extra copy of sanU and sanV was constructed and introduced into wild-type strain. HPLC results showed that nikkomycin production of recombinant strain was about 1.8 fold than that of wild-type strain. RT-PCR analysis indicated that the transcriptional level of sanU and sanV in this recombinant strain was about two folds than that of wild-type strain. The sanU and sanV were expressed in E. coli BL21 (DE3). SanU and SanV were purified individually. SanU and SanV assembled with coenzyme B12 to form a complete enzyme in vitro, which showed glutamate mutase activity. The glutamate mutase converted l-glutamate to l-threo--Methylaspartic acid, and then l-threo--Methylaspartic acid was probably deaminated to form 2-oxo-3-methylsuccinic acid to join biosynthetic pathway of the peptidyl moiety HPHT in S. ansochromogenes. SanU is the coenzyme B12-binding component and more than two folds of SanU are required for maximal enzyme activity. The optimal pH and temperature for the formed enzyme are 7.5–8.5 and 35–42°C, respectively. Sulfhydryl compounds are important for activity of the reassembled enzyme. [Copyright &y& Elsevier]

Background. Hepatocellular carcinoma (HCC) is a common cancer with an extremely high mortality rate. Therefore, there is an urgent need in screening key biomarkers of HCC to predict the prognosis and develop more individual treatments. Recently, AATF is reported to be an important factor contributing to HCC. Methods. We aimed to establish a gene signature to predict overall survival of HCC patients. Firstly, we examined the expression level of AATF in the Gene Expression Omnibus (GEO), the Cancer Genome Atlas (TCGA), and the International Union of Cancer Genome (ICGC) databases. Genes coexpressed with AATF were identified in the TCGA dataset by the Poisson correlation coefficient and used to establish a gene signature for survival prediction. The prognostic significance of this gene signature was then validated in the ICGC dataset and used to build a combined prognostic model for clinical practice. Results. Gene expression data and clinical information of 2521 HCC patients were downloaded from three public databases. AATF expression in HCC tissue was higher than that in matched normal liver tissues. 644 genes coexpressed with AATF were identified by the Poisson correlation coefficient and used to establish a three-gene signature (KIF20A, UCK2, and SLC41A3) by the univariate and multivariate least absolute shrinkage and selection operator Cox regression analyses. This three-gene signature was then used to build a combined nomogram for clinical practice. Conclusion. This integrated nomogram based on the three-gene signature can predict overall survival for HCC patients well. The three-gene signature may be a potential therapeutic target in HCC. [ABSTRACT FROM AUTHOR]

FOXA1 functions as a pioneer factor of transcriptional regulation that binds to specific sites in the chromatin and recruits other transcription factors, promoting the initiation of gene transcription and mediating the regulation of downstream target gene expression. FOXA1 was reported to facilitate or reprogram ERα binding, thus playing a key function in breast cancer progression. Our previous results indicated that the O-linked N-acetylgalactosamine (O-GalNAc) modification of FOXA1 plays a potentially significant role in the ERα transcription network. However, further investigations are needed to identify the specific mechanism of modification and the specific glycosylation sites on FOXA1. In this study, we first suggested that FOXA1 could be O-GalNAcylated by ppGalNAc-T2 in vitro. By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1, both located at the C-terminal of the protein. Then, synthesized peptides, including the predicted O-GalNAc sites in the C-terminus of FOXA1, were used in a vitro reaction, and peptides mutated at the predicted O-GalNAc sites were employed as controls. Through an ESI-MS assay, S354 and S355 were identified as probable O-GalNAcylation sites on FOXA1. Additionally, we performed ESI-ETD-MS/MS analysis of the full-length O-GalNAcylated FOXA1 protein and identified S355 as the O-GalNAc modification site on FOXA1, consistent with the peptide reaction. In conclusion, our results demonstrated that FOXA1 can be O-GalNAcylated by ppGalNAc-T2 at S355 in vitro. These results will provide new insights for studying the role of O-GalNAcylation in the development of breast cancer. • Nuclear transcription factor FOXA1, which plays a key function in tumor progression was confirmed to be O-GalNAcylated by ppGalNAc-T2 in vitro. • By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1 by HPLC and mass ESI-MS, both located at the C-terminal of the protein. • ESI-ETD-MS/MS analysis of the full-length in vitro O-GalNAcylated FOXA1 protein identified S355 as the O-GalNAc modification site on FOXA1. [ABSTRACT FROM AUTHOR]

The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1) and exoglucanase (Gux1), in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd) and Gux1 (Gux1cd), as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion. [ABSTRACT FROM AUTHOR]

Abstract: Our previous work showed that baculovirus (Antheraea pernyi nuclear polyhedrosis virus [ApNPV]) induced a strong innate immunity and protected chicken from a lethal challenge of bronchitis virus. The purpose of present study was to determine the chicken Toll-like receptors (TLRs) used by BV-infected immune cells to induce this immune response. We first investigated the expression of the Toll-like receptor (TLR) genes in resting and BV-infected HD11 (chicken macrophage-like cell line) and DT40 (chicken B cell-like cell line) cells. Expressions of TLRs were detected in both cell types. After BV stimulation, TLR21 was the only upregulated TLR in HD11 cells, and all the TLRs were down-regulated in DT40 cells. Since TLR activation generally leads to cytokine induction, we then determined the expression of IL-12 in the two cell lines following treatment with BV or oligodeoxynucleotides (ODN) containing CpG dinucleotides (CpG-ODN). BV and CpG-ODN treatment induced the expression of IL-12 in HD11 but not in DT40 cells. HD11 cells transfected with siRNA specific for TLR21 significantly diminished BV- and CpG-ODN-induced IL-12 expression. Therefore, BV and CpG-ODN stimulated IL-12 expression involved TLR21 signaling and chicken TLR21 may have similar functions to the mammalian TLR9. [Copyright &y& Elsevier]

Abstract: The budded virus (BV) of Antheraea pernyi is a nuclear polyhedrosis virus (ApNPV), that belongs to the baculovirus family. It is capable of stimulating innate immune response in avian macrophage-like cells (HD11). In this study, we used the expression of proinflammatory cytokines (IFN-γ and IL-12p40) and production of nitric oxide (NO) as indicators to evaluate the involvement of the signaling cascades in the activation of HD11 cells by BV. Western blot analysis revealed that ERK1/2, p38 MAPK and JNK were phosphorylated followed by activation of p65-NF-κB after HD11 cells were challenged with BV. Inhibition of p38 MAPK and NF-κB by their respective inhibitors abolished BV-induced expression of IL-12p40, IFN-γ and production of NO, whereas inhibition of JNK abolished only the induction of cytokines. Additionally, inhibition of ERK1/2 resulted in unexpected increases in both cytokine expression and NO production. Taken together, these findings indicated that in BV-induced HD11 cells, the induction of cytokines was mediated by the p38 MAPK, JNK and NF-κB pathways, and NO production was mediated through the p38 MAPK/NF-κB pathways. [Copyright &y& Elsevier]

Chromatin modifier metastasis-associated protein 1 (MTA1), closely associated with tumor angiogenesis in breast cancer, plays an important role in gene expression and cancer cell behavior. Recently, an association between O-GlcNAc transferase (OGT) and MTA1 was identified by mass spectroscopy. However, the potential relationship between MTA1 and O-GlcNAc modification has not yet explored. In the current study, the role of MTA1 and its O-GlcNAc modification in breast cancer cell genotoxic adaptation was investigated through quantitative proteomics, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome analysis, and loss- and gain-of-function experiments. We demonstrate that the O-GlcNAc modification promotes MTA1 to interaction with chromatin and thus changes the expression of target genes, contributing to breast cancer cell genotoxic adaptation. MTA1 is modified with O-GlcNAc residues at serine (S) residues S237/S241/S246 in adriamycin-adaptive breast cancer cells, and this modification improves the genome-wide interactions of MTA1 with gene promotor regions by enhancing its association with nucleosome remodeling and histone deacetylation (NuRD) complex. Further, O-GlcNAc modification modulates MTA1 chromatin binding, influencing the specific transcriptional regulation of genes involved in the adaptation of breast cancer cells to genotoxic stress. Our findings reveal a previously unrecognized role for O-GlcNAc-modified MTA1 in transcriptional regulation and suggest that the O-GlcNAc modification is a key to the molecular regulation of chemoresistance in breast cancers. • OGT interacts with MTA1 in breast cancer cells and promotes MTA1 O-GlcNAc modification at serine residues S237/S241/S246. • O-GlcNAc modification enhances the interaction between MTA1 and other NuRD components. • O-GlcNAc modification of MTA1 affect downstream genes expression and protect breast cancer cells against genotoxicity. [ABSTRACT FROM AUTHOR]

Background: Type III polyketide synthases (PKSs) are simple homodimer ketosynthases that distribute across plants, fungi, and bacteria, catalyzing formation of pyrone- and resorcinol-types aromatic polyketides with various bioactivities. The broad substrate promiscuity displayed by type III PKSs makes them wonderful candidates for expanding chemical diversity of polyketides. Results: Violapyrone B (VLP B, 10), an α-pyrone compound produced by deepsea-derived Streptomyces somaliensis SCSIO ZH66, is encoded by a type III PKS VioA. We overexpressed VioA in three different hosts, including Streptomyces coelicolor M1146, Streptomyces sanyensis FMA as well as the native producer S. somaliensis SCSIO ZH66, leading to accumulation of different violapyrone compounds. Among them, S. coelicolor M1146 served as the host producing the most abundant violapyrones, from which five new (2–4, 7 and 12) and nine known (1, 5, 6, 8–11, 13 and 14) compounds were identified. Anti-influenza A (H1N1) virus activity of these compounds was then evaluated using ribavirin as a positive control (IC50 = 112.9 μM), revealing that compounds 11–14 showed considerable activity with IC50 values of 112.7, 26.9, 106.7 and 28.8 μM, respectively, which are significantly improved as compared to that of VLP B (10) (IC50 > 200 μM). The productions of 10 and 13 were increased by adding P450 inhibitor metyrapone. In addition, site-directed mutagenesis experiment led to demonstration of the residue S242 to be essential for the activity of VioA. Conclusions: Biological background of the expression hosts is an important factor impacting on the encoding products of type III PKSs. By using S. coelicolor M1146 as cell factory, we were able to generate fourteen VLPs compounds. Anti-H1N1 activity assay suggested that the lipophilic nature of the alkyl chains of VLPs plays an important role for the activity, providing valuable guidance for further structural optimization of VLPs. [ABSTRACT FROM AUTHOR]