Housekeeping protein-coding genes interrogated with tissue and individual variations.

Housekeeping protein-coding genes are stably expressed genes in cells and tissues that are thought to be engaged in fundamental cellular biological functions. They are often utilized as normalization references in molecular biology research and are especially important in integrated bioinformatic investigations. Prior studies have examined human housekeeping protein-coding genes by analyzing various gene expression datasets. The inclusion of different tissue types significantly impacted the discovery of housekeeping genes. In this report, we investigated particularly individual human subject expression differences in protein-coding genes across different tissue types. We used GTEx V8 gene expression datasets obtained from more than 16,000 human normal tissue samples. Furthermore, the Gini index is utilized to investigate the expression variations of protein-coding genes between tissue and individual donor subjects. Housekeeping protein-coding genes found using Gini index profiles may vary depending on the tissue subtypes investigated, particularly given the diverse sample size collections across the GTEx tissue subtypes. We subsequently selected major tissues and identified subsets of housekeeping genes with stable expression levels among human donors within those tissues. In this work, we provide alternative sets of housekeeping protein-coding genes that show more consistent expression patterns in human subjects across major solid organs. Weblink: https://hpsv.ibms.sinica.edu.tw .

Dominant transcript expression profiles of human protein-coding genes interrogated with GTEx dataset.

The discovery and quantification of mRNA transcripts using short-read next-generation sequencing (NGS) data is a complicated task. There are far more alternative mRNA transcripts expressed by human genes than can be identified from NGS transcriptome data and various bioinformatic pipelines, while the numbers of annotated human protein-coding genes has gradually declined in recent years. It is essential to learn more about the thorough tissue expression profiles of alternative transcripts in order to obtain their molecular modulations and actual functional significance. In this report, we present a bioinformatic database for interrogating the representative tissue of human protein-coding transcripts. The database allows researchers to visually explore the top-ranked transcript expression profiles in particular tissue types. Most transcripts of protein-coding genes were found to have certain tissue expression patterns. This observation demonstrated that many alternative transcripts were particularly modulated in different cell types. This user-friendly tool visually represents transcript expression profiles in a tissue-specific manner. Identification of tissue specific protein-coding genes and transcripts is a substantial advance towards interpreting their biological functions and further functional genomics studies.

Top-ranked expressed gene transcripts of human protein-coding genes investigated with GTEx dataset.

With considerable accumulation of RNA-Seq transcriptome data, we have extended our understanding about protein-coding gene transcript compositions. However, alternatively compounded patterns of human protein-coding gene transcripts would complicate gene expression data processing and interpretation. It is essential to exhaustively interrogate complex mRNA isoforms of protein-coding genes with an unified data resource. In order to investigate representative mRNA transcript isoforms to be utilized as transcriptome analysis references, we utilized GTEx data to establish a top-ranked transcript isoform expression data resource for human protein-coding genes. Distinctive tissue specific expression profiles and modulations could be observed for individual top-ranked transcripts of protein-coding genes. Protein-coding transcripts or genes do occupy much higher expression fraction in transcriptome data. In addition, top-ranked transcripts are the dominantly expressed ones in various normal tissues. Intriguingly, some of the top-ranked transcripts are noncoding splicing isoforms, which imply diverse gene regulation mechanisms. Comprehensive investigation on the tissue expression patterns of top-ranked transcript isoforms is crucial. Thus, we established a web tool to examine top-ranked transcript isoforms in various human normal tissue types, which provides concise transcript information and easy-to-use graphical user interfaces. Investigation of top-ranked transcript isoforms would contribute understanding on the functional significance of distinctive alternatively spliced transcript isoforms.

miR-TV: an interactive microRNA Target Viewer for microRNA and target gene expression interrogation for human cancer studies.

MicroRNAs (miRNAs) have been identified in many organisms, and they are essential for gene expression regulation in many critical cellular processes. The expression levels of these genes and miRNAs are closely associated with the progression of diseases such as cancers. Furthermore, survival analysis is a significant indicator for evaluating the criticality of these cellular processes in cancer progression. We established a web tool, miRNA Target Viewer (miR-TV), which integrates 5p-arm and 3p-arm miRNA expression profiles, mRNA target gene expression levels in healthy and cancer populations, and clinical data of cancer patients and their survival information. The developed miR-TV obtained miRNA-seq, mRNA-seq and clinical data from the Cancer Genome Atlas and potential miRNA target gene predictions from miRDB, targetScan and miRanda. The data presentation was implemented using the D3 javascript toolkit. The D3 toolkit is frequently used to provide an easy-to-use interactive interface. Our miR-TV provides a user-friendly and interactive interface, which can be beneficial for biomedical researchers to freely interrogate miRNA expression information and their potential target genes. We believe that such a data visualization bioinformatics tool is excellent for obtaining information from massive biological data. Database URL: http://mirtv.ibms.sinica.edu.tw.

MiR-182-5p enhances in vitro neutrophil infiltration in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) patients could develop coronary artery lesion (CAL) which threatens children's life. A previous study identified KD biomarker miRNAs that could discriminate KD patients from febrile non-KD patients. We wonder whether these KD prediction biomarkers could be further applied to predict CAL formation in KD patients. METHODS: To examine this hypothesis, we conducted a meta-analysis, miRNA mimic transfection, in vitro cell model and microarray assays. RESULTS: We first showed that miR-182-5p and miR-183-5p kept higher levels in the KD patients with CAL than those without CAL (p < .05). Further machine learning alignment confirmed that CAL formation could be predicted, with an auROC value of 0.86. We further treated neutrophil cells with miR-182-5p mimic, followed by in vitro transendotherial migration assay. As a result, miR-182-5p overexpression significantly (p < .05) enhanced neutrophil cells to infiltrate the endothelial layer composed of human coronary artery endothelium cells. Further microarray assay and pathway enrichment analysis showed that the genes activated with miR-182-5p overexpression were significantly enriched in the leukocyte transendothelial migration pathway (kegg_pathway_194, p < .05). CONCLUSION: Therefore, our study suggested that miR-182-5p enhanced in vitro leukocyte infiltration by activating the leukocyte transendothelial migration pathway in CAL formation in KD.

Overlapping protein-coding genes in human genome and their coincidental expression in tissues.

The completion of human genome sequences and the advancement of next-generation sequencing technologies have engendered a clear understanding of all human genes. Overlapping genes are usually observed in compact genomes, such as those of bacteria and viruses. Notably, overlapping protein-coding genes do exist in human genome sequences. Accordingly, we used the current Ensembl gene annotations to identify overlapping human protein-coding genes. We analysed 19,200 well-annotated protein-coding genes and determined that 4,951 protein-coding genes overlapped with their adjacent genes. Approximately a quarter of all human protein-coding genes were overlapping genes. We observed different clusters of overlapping protein-coding genes, ranging from two genes (paired overlapping genes) to 22 genes. We also divided the paired overlapping protein-coding gene groups into four subtypes. We found that the divergent overlapping gene subtype had a stronger expression association than did the subtypes of 5'-tandem overlapping and 3'-tandem overlapping genes. The majority of paired overlapping genes exhibited comparable coincidental tissue expression profiles; however, a few overlapping gene pairs displayed distinctive tissue expression association patterns. In summary, we have carefully examined the genomic features and distributions about human overlapping protein-coding genes and found coincidental expression in tissues for most overlapping protein-coding genes.

Short-term and long-term impacts of Helicobacter pylori eradication with reverse hybrid therapy on the gut microbiota.

BACKGROUND AND AIMS: Anti-Helicobacter pylori therapy may lead to the growth of pathogenic or antibiotic-resistant bacteria in the gut. The study aimed to investigate the short-term and long-term impacts of H. pylori eradication with reverse hybrid therapy on the components and macrolide resistance of the gut microbiota. METHODS: Helicobacter pylori-related gastritis patients were administered a 14-day reverse hybrid therapy. Fecal samples were collected before treatment and at the end of week 2, week 8, and week 48. The V3-V4 region of the bacterial 16S rRNA gene in fecal specimens was amplified by polymerase chain reaction and sequenced on Illumina MiSeq platform. Additionally, amplification of erm(B) gene (encoding erythromycin resistance methylase) was performed. RESULTS: Reverse hybrid therapy resulted in decreased relative abundances of Firmicutes (from 62.0% to 30.7%; P < 0.001) and Actinobacteria (from 3.4% to 0.6%; 0.032) at the end of therapy. In contrast, the relative abundance of Proteobacteria increased from 10.2% to 49.1% (0.002). These microbiota alterations did not persist but returned to the initial levels at week 8 and week 48. The amount of erm(B) gene in fecal specimens was comparable with the pretreatment level at week 2 but increased at week 8 (0.025) and then returned to the pretreatment level by week 48. CONCLUSIONS: Helicobacter pylori eradication with reverse hybrid therapy can lead to short-term gut dysbiosis. The amount of erm(B) gene in the stool increased transiently after treatment and returned to the pretreatment level at 1-year post-treatment.

Helicobacter pylori eradication with bismuth quadruple therapy leads to dysbiosis of gut microbiota with an increased relative abundance of Proteobacteria and decreased relative abundances of Bacteroidetes and Actinobacteria.

BACKGROUND: Bismuth quadruple therapy is the treatment of choice for the first-line therapy of Helicobacter pylori infection in areas of high clarithromycin resistance. Currently, the impact of the promising treatment on gut microbiota remains unclear. AIM: To investigate the short-term and long-term impacts of bismuth quadruple therapy on gut microbiota. METHODS: Adult patients with H. pylori-related gastritis were treated with 14-day bismuth quadruple therapy. Fecal samples were collected before treatment at week 2, week 8, and week 48. Nucleic acid extraction from fecal samples was performed. The V3-V4 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction and sequenced with the MiSeq followed by data analysis using Qiime pipeline. RESULTS: Eleven patients received complete follow-up. Before treatment, the most abundant phyla were Firmicutes (45.3%), Bacteroidetes (24.3%), Proteobacteria (9.9%), and Actinobacteria (5.0%). At the end of bismuth therapy, the relative abundances of Bacteroidetes and Actinobacteria decreased to 0.5% (P < .001) and 1.3% (P = .038), respectively. Additionally, the relative abundance of Verrucomicrobia also decreased from 3.2% to 1.11E-3% (P = .034). In contrast, the relative abundances of Proteobacteria and Cyanobacteria increased (P < .001 and P = .003, respectively). At week 8, the relative abundances of all phyla restored to the levels at baseline. The relative abundances of all phyla at week 48 also did not significantly differ from those at baseline. During eradication therapy, 6 patients (55%) reported at least 1 adverse event. The relative abundance of phylum Proteobacteria in patients with adverse effects was more than that in patients without adverse effects (68.7% ± 8.8% vs 43.4% ± 25.5%; P = .048). CONCLUSIONS: Bismuth quadruple therapy for H. pylori eradication can lead to short-term dysbiosis of gut microbiota. The increase in Proteobacteria in gut microbiota may attribute to the development of adverse effects during bismuth quadruple therapy.

Urine miR-21-5p as a potential non-invasive biomarker for gastric cancer.

Many reports have implicated that microRNAs involve in cancer development and progression, such as miR-155 in breast cancers and miR-196 in gastric cancers. Furthermore, microRNAs are more stable than typical protein-coding gene mRNAs in varieties of clinical samples including body fluids. This suggests that they are potentially valuable biomarkers for cancer monitoring. In this study, we have used urine samples of gastric cancer patients to demonstrate the feasibility of urine microRNAs for gastric cancer detection. Urine samples of gastric cancer patients were extracted for total RNA, which were examined for the expression of miR-21-5p using quantitative stem-loop PCR. Our results demonstrated that miR-21-5p could be detected in small amounts of urine samples with good stability, and the expression levels of miR-21-5p were reduced following surgical removal of gastric cancer tissues. These results implicate that urine miR-21-5p could be utilized as a novel non-invasive biomarker of gastric cancer detection and monitoring.

Visual Display of 5p-arm and 3p-arm miRNA Expression with a Mobile Application.

MicroRNAs (miRNAs) play important roles in human cancers. In previous studies, we have demonstrated that both 5p-arm and 3p-arm of mature miRNAs could be expressed from the same precursor and we further interrogated the 5p-arm and 3p-arm miRNA expression with a comprehensive arm feature annotation list. To assist biologists to visualize the differential 5p-arm and 3p-arm miRNA expression patterns, we utilized a user-friendly mobile App to display. The Cancer Genome Atlas (TCGA) miRNA-Seq expression information. We have collected over 4,500 miRNA-Seq datasets from 15 TCGA cancer types and further processed them with the 5p-arm and 3p-arm annotation analysis pipeline. In order to be displayed with the RNA-Seq Viewer App, annotated 5p-arm and 3p-arm miRNA expression information and miRNA gene loci information were converted into SQLite tables. In this distinct application, for any given miRNA gene, 5p-arm miRNA is illustrated on the top of chromosome ideogram and 3p-arm miRNA is illustrated on the bottom of chromosome ideogram. Users can then easily interrogate the differentially 5p-arm/3p-arm expressed miRNAs with their mobile devices. This study demonstrates the feasibility and utility of RNA-Seq Viewer App in addition to mRNA-Seq data visualization.

miRSeq: a user-friendly standalone toolkit for sequencing quality evaluation and miRNA profiling.

MicroRNAs (miRNAs) present diverse regulatory functions in a wide range of biological activities. Studies on miRNA functions generally depend on determining miRNA expression profiles between libraries by using a next-generation sequencing (NGS) platform. Currently, several online web services are developed to provide small RNA NGS data analysis. However, the submission of large amounts of NGS data, conversion of data format, and limited availability of species bring problems. In this study, we developed miRSeq to provide alternatives. To test the performance, we had small RNA NGS data from four species, including human, rat, fly, and nematode, analyzed with miRSeq. The alignments results indicate that miRSeq can precisely evaluate the sequencing quality of samples regarding percentage of self-ligation read, read length distribution, and read category. miRSeq is a user-friendly standalone toolkit featuring a graphical user interface (GUI). After a simple installation, users can easily operate miRSeq on a PC or laptop by using a mouse. Within minutes, miRSeq yields useful miRNA data, including miRNA expression profiles, 3' end modification patterns, and isomiR forms. Moreover, miRSeq supports the analysis of up to 105 animal species, providing higher flexibility.

The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1.

The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

Coexpression-based clustering of Arabidopsis root genes predicts functional modules in early phosphate deficiency signaling.

Phosphate (Pi) deficiency triggers the differential expression of a large set of genes, which communally adapt the plant to low Pi bioavailability. To infer functional modules in early transcriptional responses to Pi deficiency, we conducted time-course microarray experiments and subsequent coexpression-based clustering of Pi-responsive genes by pairwise comparison of genes against a customized database. Three major clusters, enriched in genes putatively functioning in transcriptional regulation, root hair formation, and developmental adaptations, were predicted from this analysis. Validation of gene expression by quantitative reverse transcription-PCR revealed that transcripts from randomly selected genes were robustly induced within the first hour after transfer to Pi-deplete medium. Pectin-related processes were among the earliest and most robust responses to Pi deficiency, indicating that cell wall alterations are critical in the early transcriptional response to Pi deficiency. Phenotypical analysis of homozygous mutants defective in the expression of genes from the root hair cluster revealed eight novel genes involved in Pi deficiency-induced root hair elongation. The plants responded rapidly to Pi deficiency by the induction of a subset of transcription factors, followed by a repression of genes involved in cell wall alterations. The combined results provide a novel, integrated view at a systems level of the root responses that acclimate Arabidopsis (Arabidopsis thaliana) to suboptimal Pi levels.

Bioinformatic discovery of microRNA precursors from human ESTs and introns.

BACKGROUND: MicroRNAs (miRNAs) function in many physiological processes, and their discovery is beneficial for further studying their physiological functions. However, many of the miRNAs predicted from genomic sequences have not been experimentally validated to be authentic expressed RNA transcripts, thereby decreasing the reliability of miRNA discovery. To overcome this problem, we examined expressed transcripts - ESTs and intronic sequences - to identify novel miRNAs as well as their target genes. RESULTS: To facilitate our approach, we developed our scanning method using criteria based on the features of 207 known human pre-miRNAs to discriminate miRNAs from random sequences. We identified 208 candidate hairpins in human ESTs and human reference gene intronic sequences, 52 of which are known pre-miRNAs. The discovery pipeline performance was further assessed using 130 newly updated pre-miRNA and randomly selected sequences. We achieved sensitivity of 85% (110/130) and overall specificity of 49.7% using this method. Because miRNAs are evolutionarily conserved regulators of gene expression, it is expected that their host genes and target genes should have respective phylogenetic orthologs. Our results confirmed that, in certain mammals, the host genes carrying the same miRNAs are orthologs, as previously reported. Moreover, this observation is also the case for some of the miRNA target genes. CONCLUSION: We have predicted 208 human pre-miRNA candidates and over 10,000 putative human target genes. Using sequence information from ESTs and introns ensures that the predicted pre-miRNA candidates are expressed and the combined expression transcription information from ESTs and introns makes our prediction results more decisive with regard to expressed pre-miRNAs.