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Recently, we showed that three months of resistance exercise significantly alters 18 canonical pathways related to chronic inflammation in PBMCs of older adults. In this exploratory sub-study, the aim is to explore whether resistance exercise enhances the PBMCs stress response by mimicking an acute infection through in vitro LPS stimulation. Women (≥65 years) were randomly divided into intensive strength training (IST), strength endurance training (SET), or flexibility training (as control group, CON) groups. PBMCs were isolated and cultured with and without LPS for 24 h. Their RNA was analyzed via targeted RNA sequencing of 407 inflammation-related genes, with relevant fold-changes defined as ≤0.67 or ≥1.5 (3 months vs. baseline). A pathway analysis using ingenuity pathway analyses identified significant pathways among 407 genes with p < 0.05 and z-scores of ≤−2 or ≥2. Fourteen women were included in the analyses. A total of 151 genes with a significant fold-change were identified. In the CON group, a less-pronounced effect was observed. Strength training altered 23 pathways in the LPS-stimulated PBMCs, none of which overlapped between the IST and SET groups. A balanced exercise program that includes both IST and SET could beneficially adapt the immune responses in older adults by inducing alterations in the inflammatory stress response of PBMCs through different genes and pathways. [ABSTRACT FROM AUTHOR]

Abstract Hepatitis C virus (HCV) infection poses a significant public health challenge and often leads to long-term health complications and even death. Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a proposed viral etiology. HCV infection and PD have been previously suggested to be related. This work aimed to identify potential biomarkers and pathways that may play a role in the joint development of PD and HCV infection. Using BioOptimatics-bioinformatics driven by mathematical global optimization-, 22 publicly available microarray and RNAseq datasets for both diseases were analyzed, focusing on sex-specific differences. Our results revealed that 19 genes, including MT1H, MYOM2, and RPL18, exhibited significant changes in expression in both diseases. Pathway and network analyses stratified by sex indicated that these gene expression changes were enriched in processes related to immune response regulation in females and immune cell activation in males. These findings suggest a potential link between HCV infection and PD, highlighting the importance of further investigation into the underlying mechanisms and potential therapeutic targets involved.

Recently, we showed that three months of resistance exercise significantly alters 18 canonical pathways related to chronic inflammation in PBMCs of older adults. In this exploratory sub-study, the aim is to explore whether resistance exercise enhances the PBMCs stress response by mimicking an acute infection through in vitro LPS stimulation. Women (≥65 years) were randomly divided into intensive strength training (IST), strength endurance training (SET), or flexibility training (as control group, CON) groups. PBMCs were isolated and cultured with and without LPS for 24 h. Their RNA was analyzed via targeted RNA sequencing of 407 inflammation-related genes, with relevant fold-changes defined as ≤0.67 or ≥1.5 (3 months vs. baseline). A pathway analysis using ingenuity pathway analyses identified significant pathways among 407 genes with p < 0.05 and z-scores of ≤−2 or ≥2. Fourteen women were included in the analyses. A total of 151 genes with a significant fold-change were identified. In the CON group, a less-pronounced effect was observed. Strength training altered 23 pathways in the LPS-stimulated PBMCs, none of which overlapped between the IST and SET groups. A balanced exercise program that includes both IST and SET could beneficially adapt the immune responses in older adults by inducing alterations in the inflammatory stress response of PBMCs through different genes and pathways.

In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume.

The mammary glands, responsible for milk secretion, are regulated at a local level by various hormones, growth factors, non-coding RNAs, and other elements. Recent research has discovered the presence of lncRNAs in these glands, with suggestions that they may be essential for the maintenance and function of mammary glands. Besides directly controlling the gene and protein expression, lncRNAs are believed to play a significant part in numerous physiological and pathological processes. This study focused on examining the mammary gland tissues of Chinese Holstein cows, to identify and categorize long non-coding RNAs (lncRNAs). The research intended to distinguish lncRNAs in the mammary tissues of Holstein cows and contrast them between lactation and non-lactation periods. In this study, mammary gland tissues were sampled from three Holstein cows in early lactation (n = 3, 30 days postpartum) and non-lactation (n = 3, 315 days postpartum) on a large dairy farm in Jiangsu province. Mammary tissue samples were collected during early lactation and again during non-lactation. In total, we detected 1905 lncRNAs, with 57.3% being 500 bp and 612 intronic lncRNAs. The exon count for lncRNAs varied from 2 to 10. It was observed that 96 lncRNA expressions markedly differed between the two stages, with 83 genes being upregulated and 53 downregulated. Enrichment analysis results revealed that Gene Ontology (GO) analysis was primarily abundant in cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that target genes were predominantly abundant in metabolic pathways, fatty acid biosynthesis, the immune system, and glycosphingolipid biosynthesis. This study analyzed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows during both lactation and non-lactation stages, forming a foundation for further investigation into the functional roles of lncRNAs in Holstein cows throughout lactation. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the gene mutation, immune infiltration and tumor growth of primary tumor and distant tumor under different treatment modes. Materials and Methods: Twenty B16 murine melanoma cells were injected subcutaneously into the of both sides of the thigh, simulating a primary tumor and a secondary tumor impacted by the abscopal effect, respectively. They were divided into blank control group, immunotherapy group, radiotherapy group, and radiotherapy combined immunotherapy group. During this period, tumor volume was measured, and RNA sequencing was performed on tumor samples after the test. R software was used to analyze differentially expressed genes, functional enrichment, and immune infiltration. Results: We found that any treatment mode could cause changes in differentially expressed genes, especially the combination treatment. The different therapeutic effects might be caused by gene expression. In addition, the proportions of infiltrating immune cells in the irradiated and abscopal tumors were different. In the combination treatment group, T-cell infiltration in the irradiated site was the most obvious. In the immunotherapy group, CD8+ T-cell infiltration in the abscopal tumor site was obvious, but immunotherapy alone might have a poor prognosis. Whether the irradiated or abscopal tumor was evaluated, radiotherapy combined with anti-programmed cell death protein 1 (anti- PD-1) therapy produced the most obvious tumor control and might have a positive impact on prognosis. Conclusion: Combination therapy not only improves the immune microenvironment but may also have a positive impact on prognosis. [ABSTRACT FROM AUTHOR]

The mammary glands, responsible for milk secretion, are regulated at a local level by various hormones, growth factors, non-coding RNAs, and other elements. Recent research has discovered the presence of lncRNAs in these glands, with suggestions that they may be essential for the maintenance and function of mammary glands. Besides directly controlling the gene and protein expression, lncRNAs are believed to play a significant part in numerous physiological and pathological processes. This study focused on examining the mammary gland tissues of Chinese Holstein cows, to identify and categorize long non-coding RNAs (lncRNAs). The research intended to distinguish lncRNAs in the mammary tissues of Holstein cows and contrast them between lactation and non-lactation periods. In this study, mammary gland tissues were sampled from three Holstein cows in early lactation (n = 3, 30 days postpartum) and non-lactation (n = 3, 315 days postpartum) on a large dairy farm in Jiangsu province. Mammary tissue samples were collected during early lactation and again during non-lactation. In total, we detected 1905 lncRNAs, with 57.3% being 500 bp and 612 intronic lncRNAs. The exon count for lncRNAs varied from 2 to 10. It was observed that 96 lncRNA expressions markedly differed between the two stages, with 83 genes being upregulated and 53 downregulated. Enrichment analysis results revealed that Gene Ontology (GO) analysis was primarily abundant in cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that target genes were predominantly abundant in metabolic pathways, fatty acid biosynthesis, the immune system, and glycosphingolipid biosynthesis. This study analyzed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows during both lactation and non-lactation stages, forming a foundation for further investigation into the functional roles of lncRNAs in Holstein cows throughout lactation.

Plain English summary Patients are increasingly becoming involved in all stages of the research process. Patient and public involvement has been shown to answer questions that matter to patients, increase enrollment in studies, and improve the spread of research results to the public. However, there are limited evaluations of patient engagement in basic science research (research performed in a laboratory setting in various fields). Here, we provide an example of patients effectively involved in the planning and implementation of an ethically complex study in the field of Assistive Reproductive Technology. A Patient Engagement Studio, affiliated with an academic health center, directly connects patients and researchers through bi-monthly meetings. Recently, two researchers presented their project, Embryo+™, to the studio during the planning stage of their study. This project aims to use a new testing technology to reduce miscarriage rates. The researchers presented to the studio twice (fall 2019), and two follow-up meetings were conducted with the patients (summer 2020). Also, the patients completed a survey evaluating how engaged they felt with the project. Through the meetings, the researchers changed their language in response to patient feedback, and patients developed project protocols. Survey results showed that the patients thought this project was very meaningful and valuable. Overall, this evaluation shows that patients can add value to contentious bench science projects, particularly in the field of Assistive Reproductive Technology.

Purpose: Form deprivation myopia (FDM) is an urgent public issue characterized by pathological changes, but the underlying mechanism remained unclear. The aim was to investigate bone morphogenetic proteins (BMPs) utilizing the pathogenesis of FDM. Material and methods: Gene expression omnibus (GEO) database was used to analyze one mRNA profile (GSE89325) of FDM. Sixteen retina samples (8 FDM and 8 controls) were randomly divided into seven groups for differential gene expression analysis in R. software. The gene pathway and protein-protein interaction (PPI) analysis were performed by the DAVID and STRING databases. Cytoscape was used to draw the PPI network. The gene ontology (GO) enrichment and Kyoto encyclopedia of genes and Genomes (KEGG) analysis were determined to achieve gene annotation and visualization. Results: A total of 18420 differentially expressed genes (DEGs) were identified associated with FDM. The only non-significant gene (BEND6) was separately analyzed between two groups. Thirteen hub genes were discovered, ACVR1, ACVR2A, ACVR2B, RGMB, BMPR2, BMPR1A, BMP2, BMPR1B, CHRD, PTH, PTH1R, PTHLH, and WNT9A. The expression alteration in FDM were mainly enriched in cytokine-cytokine, and neuroactive ligand receptor interaction pathways. BMP2 was the key gene in myopia progression. Conclusions: Of clinical perspective, our findings reveal that expression of BMP2 as an underlying mechanism of FDM, providing an insight for therapeutic interventions.

Small non-coding RNAs (ncRNAs) are attracting increasing attention as they are now considered potentially valuable resources in the development of new drugs intended to cure several human diseases. A prerequisite for the development of drugs targeting ncRNAs or the related pathways is the identification and correct classification of such ncRNAs. State-of-the-art small ncRNA classification methodologies use secondary structural features as input. However, such feature extraction approaches only take global characteristics into account and completely ignore co-relative effects of local structures. Furthermore, secondary structure based approaches incorporate high dimensional feature space which is computationally expensive. The present paper proposes a novel Robust and Precise ConvNet (RPC-snRC) methodology which classifies small ncRNAs into relevant families by utilizing their primary sequence. RPC-snRC methodology learns hierarchical representation of features by utilizing positioning and information on the occurrence of nucleotides. To avoid exploding and vanishing gradient problems, we use an approach similar to DenseNet in which gradient can flow straight from subsequent layers to previous layers. In order to assess the effectiveness of deeper architectures for small ncRNA classification, we also adapted two ResNet architectures having a different number of layers. Experimental results on a benchmark small ncRNA dataset show that the proposed methodology does not only outperform existing small ncRNA classification approaches with a significant performance margin of 10% but it also gives better results than adapted ResNet architectures. To reproduce the results Source code and data set is available at https://github.com/muas16/small-non-coding-RNA-classification.

Versatile RNA modifications play important roles in post-transcriptional regulations of gene expression, among which glycosylation modifications on small RNAs emerge as a novel clade whose characteristics need further interrogations. Here, we demonstrated that the sequence pattern around RNA glycosylation sites was not random and could be exploited for glycosylation site prediction. A machine learning predictor, GlyinsRNA, which integrated multiple RNA sequence representation encodings, was established. GlyinsRNA achieved AUROC (area under the receiver operating characteristic curve) of 0.7933 and 0.7979 in five-fold cross-validation and independent tests, respectively. GlyinsRNA was implemented as an online webserver, where both the predicted glycosylation sites and the overrepresented RNA-binding protein (RBP)-related motifs were annotated to facilitate the users. GlyinsRNA webserver is freely available at . [ABSTRACT FROM AUTHOR]

Background: Patient and Public Involvement (PPI) in research is increasingly being utilized to better connect patients and researchers. The Patient Engagement Studio (PES) supports PPI in research by working directly with researchers throughout various stages of their projects. Recently, two researchers presented to the PES for assistance with their project, Embryo+™. The purpose of Embryo+™ is to decrease miscarriage rates using RNA sequencing technology that screens for the most viable embryos. To date, no examples of PPI directly in the planning or implementation of bench research concerning in vitro fertilization and embryo transfer have been identified. Main body: Embryo+™ researchers met in-person with the PES two times (fall 2019; each meeting had 9 PES members in attendance) for initial feedback and protocol development. After these meetings, PES leadership and Embryo+™ researchers decided that the unique nature of the project merited a PPI evaluation. Subsequent evaluation of engagement efforts occurred by reviewing the PES reports for the Embryo+™ researchers, conducting two recorded web-based discussion meetings with the PES (summer 2020; meeting 1 n = 7; meeting 2 n = 6), and a brief survey (n = 13). The discussion meetings provided an opportunity for the PES members to define engagement themes through consensus via verbal agreement to the studio director's periodic summaries during the discussions. Combining survey results and PES themes allowed for a broad discussion for meaningful engagement. The Embryo+™ researchers established trust with the patients by changing some of their language in response to patient suggestions, allowing for unintended ethical conversations, and implementing the patient developed protocols. Overall, the patient experts thought this project was very meaningful and valuable, quantified by a mean loyalty score 89.43 (s.d. 10.29). Conclusion: Bench science researchers may need additional PPI training prior to engaging with patient groups. PPI in this project was successful in large part due to this training, where the director emphasized the importance of gaining trust with the patients. The researchers applied what they learned and several examples of how to develop trust with patients are discussed. If trust is established, PPI in an ethically charged, basic science research study can be both valuable and successful. Plain English summary: Patients are increasingly becoming involved in all stages of the research process. Patient and public involvement has been shown to answer questions that matter to patients, increase enrollment in studies, and improve the spread of research results to the public. However, there are limited evaluations of patient engagement in basic science research (research performed in a laboratory setting in various fields). Here, we provide an example of patients effectively involved in the planning and implementation of an ethically complex study in the field of Assistive Reproductive Technology. A Patient Engagement Studio, affiliated with an academic health center, directly connects patients and researchers through bi-monthly meetings. Recently, two researchers presented their project, Embryo+™, to the studio during the planning stage of their study. This project aims to use a new testing technology to reduce miscarriage rates. The researchers presented to the studio twice (fall 2019), and two follow-up meetings were conducted with the patients (summer 2020). Also, the patients completed a survey evaluating how engaged they felt with the project. Through the meetings, the researchers changed their language in response to patient feedback, and patients developed project protocols. Survey results showed that the patients thought this project was very meaningful and valuable. Overall, this evaluation shows that patients can add value to contentious bench science projects, particularly in the field of Assistive Reproductive Technology. [ABSTRACT FROM AUTHOR]

ICH Model Larvae from heterozygous adult I bbh i Tg[ I mpo: i GFP; I mpeg1: i mcherry] (referred to as mpo:mpeg) in-crosses were collected and light microscopy used to identify brain hemorrhages (ICH+) in homozygotes, which were separated from non-hemorrhaged (ICH-) heterozygous and wild-type siblings at ~52 hpf. The secondary injury response in zebrafish larvae is identified by an increase in macrophage recruitment and activation within the head at 24 h post injury, whilst neutrophils are the most numerous leukocyte population at this developmental stage. Keywords: RNA sequence analysis; zebrafish; leukocytes; intracerebral hemorrhage; translational research EN RNA sequence analysis zebrafish leukocytes intracerebral hemorrhage translational research N.PAG N.PAG 7 05/17/21 20210414 NES 210414 Introduction The pathogenesis of intracerebral hemorrhage (ICH) can be divided into two distinct injury phases. [Extracted from the article]

Neonatal sepsis (NS) kills 750,000 infants every year. Effectively treating NS requires timely diagnosis and antimicrobial therapy matched to the causative pathogens, but most blood cultures for suspected NS do not recover a causative pathogen. We refer to these suspected but unidentified pathogens as microbial dark matter. Given these low culture recovery rates, many non-culture-based technologies are being explored to diagnose NS, including PCR, 16S amplicon sequencing, and whole metagenomic sequencing. However, few of these newer technologies are scalable or sustainable globally. To reduce worldwide deaths from NS, one possibility may be performing population-wide pathogen discovery. Because pathogen transmission patterns can vary across space and time, computational models can be built to predict the pathogens responsible for NS by region and season. This approach could help to optimally treat patients, decreasing deaths from NS and increasing antimicrobial stewardship until effective diagnostics that are scalable become available globally. [ABSTRACT FROM AUTHOR]

Potential pathogenic factors, other than well-known APP, APOE4, and PSEN, can be further identified from transcriptomics studies of differentially expressed genes (DEGs) that are specific for Alzheimer’s disease (AD), but findings are often inconsistent or even contradictory. Evidence corroboration by combining meta-analysis and bioinformatics methods may help to resolve existing inconsistencies and contradictions. This study aimed to demonstrate a systematic workflow for evidence synthesis of transcriptomic studies using both meta-analysis and bioinformatics methods to identify potential pathogenic factors. Transcriptomic data were assessed from GEO and ArrayExpress after systematic searches. The DEGs and their dysregulation states from both DNA microarray and RNA sequencing datasets were analyzed and corroborated by meta-analysis. Statistically significant DEGs were used for enrichment analysis based on KEGG and protein–protein interaction network (PPIN) analysis based on STRING. AD-specific modules were further determined by the DIAMOnD algorithm, which identifies significant connectivity patterns between specific disease-associated proteins and non-specific proteins. Within AD-specific modules, the nodes of highest degrees (>95th percentile) were considered as potential pathogenic factors. After systematic searches of 225 datasets, extensive meta-analyses among 25 datasets (21 DNA microarray datasets and 4 RNA sequencing datasets) identified 9,298 DEGs. The dysregulated genes and pathways in AD were associated with impaired amyloid-β (Aβ) clearance. From the AD-specific module, Fyn, and EGFR were the most statistically significant and biologically relevant. This meta-analytical study suggested that the reduced Aβ clearance in AD pathogenesis was associated with the genes encoding Fyn and EGFR, which were key receptors in Aβ downstream signaling.

Stress-associated proteins (SAPs) are zinc finger proteins involved in the regulation of various stresses in a variety of plant species. A total of nine PdSAP genes were identified in Prunus dulcis. Phylogenetic and synteny analyses were performed to analyze the homology and evolutionary relationship of PdSAP genes. The functions of PdSAP genes were assessed by further analyses, including cis-regulatory elements, gene duplication, gene ontology, gene structure, subcellular localization, and motif pattern. This study found that PdSAP genes were unevenly distributed on chromosomes 2, 3, 6, and 7. Phylogenetic analysis of PdSAP genes with Arabidopsis thaliana and Oryza sativa suggested that six subgroups have a similar pattern of AN1 and A20 domains in each subgroup. PdSAP genes lacked duplicated blocks. The majority of PdSAP genes were localized in the nucleus region. Three hormonal and five stress cis-regulatory elements were found in the upstream promoter region of the PdSAP gene family. RNA-seq analysis revealed differential gene expression of PdSAP genes at days 12, 17, 22, 27, 32, and 37 of fruitlet development after flowering. This study identifies the SAP genes in P. dulcis and also provides insights into the expression of PdSAP genes in abnormal fruitlets with diapause atrophic growth at various developmental stages.

Potential pathogenic factors, other than well-known APP , APOE4 , and PSEN , can be further identified from transcriptomics studies of differentially expressed genes (DEGs) that are specific for Alzheimer's disease (AD), but findings are often inconsistent or even contradictory. Evidence corroboration by combining meta-analysis and bioinformatics methods may help to resolve existing inconsistencies and contradictions. This study aimed to demonstrate a systematic workflow for evidence synthesis of transcriptomic studies using both meta-analysis and bioinformatics methods to identify potential pathogenic factors. Transcriptomic data were assessed from GEO and ArrayExpress after systematic searches. The DEGs and their dysregulation states from both DNA microarray and RNA sequencing datasets were analyzed and corroborated by meta-analysis. Statistically significant DEGs were used for enrichment analysis based on KEGG and protein–protein interaction network (PPIN) analysis based on STRING. AD-specific modules were further determined by the DIAMOnD algorithm, which identifies significant connectivity patterns between specific disease-associated proteins and non-specific proteins. Within AD-specific modules, the nodes of highest degrees (>95th percentile) were considered as potential pathogenic factors. After systematic searches of 225 datasets, extensive meta-analyses among 25 datasets (21 DNA microarray datasets and 4 RNA sequencing datasets) identified 9,298 DEGs. The dysregulated genes and pathways in AD were associated with impaired amyloid-β (Aβ) clearance. From the AD-specific module, Fyn, and EGFR were the most statistically significant and biologically relevant. This meta-analytical study suggested that the reduced Aβ clearance in AD pathogenesis was associated with the genes encoding Fyn and EGFR, which were key receptors in Aβ downstream signaling. [ABSTRACT FROM AUTHOR]

In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume. [Display omitted] • AON treatment induced about 40% exon skip and Htt protein modification in cortex. • AON treatment rescued the YAC128 phenotype on body weight and activity levels. • The gene expression signature in YAC128 striatum was ameliorated by AON treatment. • MRI showed that AON treatment improved striatal volume. • Treatment with scrambled AON also affected the HD gene signature and brain volume. [ABSTRACT FROM AUTHOR]

Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca2+-currents, likely responsible for fusion and/or fission events of endolysosomal membranes and thereby for intracellular vesicle trafficking. Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis.

mTORC1 signaling plays an important role in extracellular and intracellular signals, including growth factors, nutrients, energy metabolism, and stress. However, the functional role of mTORC1 in dentinogenesis is unknown. To study the role of Raptor/mTORC1 in dentinogenesis, an Raptorfl/fl; Osx-Cre (Rap-Osx) mouse, in which Raptor was conditionally deleted in odontoblasts and dental mesenchymal cells, was generated, and postnatal tooth development was compared between Rap-Osx mice and control littermates. Rap-Osx mice presented a phenotype known as dentinogenesis imperfecta and had smaller tooth volume, a thinner dentin layer and a larger pulp chamber. The proliferation and differentiation of odontoblasts/preodontoblasts were attenuated in mutant mice, which was likely responsible for the defects in dentinogenesis. Raptor/mTORC1-pS6K1 signaling was inactivated during tooth development in Rap-Osx mice, whereas it was activated in control mice. These results indicate that Raptor/mTORC1 plays a critical role in dentinogenesis via promoting odontoblasts/preodontoblasts proliferation and differentiation. Raptor/mTORC1 might regulate tooth development through the pS6K1 signaling pathway.

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis. [ABSTRACT FROM AUTHOR]

While sequencing ancient DNA (aDNA) from archaeological material is now commonplace, very few attempts to sequence ancient transcriptomes have been made, even from typically stable deposition environments such as permafrost. This is presumably due to assumptions that RNA completely degrades relatively quickly, particularly when dealing with autolytic, nuclease-rich mammalian tissues. However, given the recent successes in sequencing ancient RNA (aRNA) from various sources including plants and animals, we suspect that these assumptions may be incorrect or exaggerated. To challenge the underlying dogma, we generated shotgun RNA data from sources that might normally be dismissed for such study. Here, we present aRNA data generated from two historical wolf skins, and permafrost-preserved liver tissue of a 14,300-year-old Pleistocene canid. Not only is the latter the oldest RNA ever to be sequenced, but it also shows evidence of biologically relevant tissue specificity and close similarity to equivalent data derived from modern-day control tissue. Other hallmarks of RNA sequencing (RNA-seq) data such as exon-exon junction presence and high endogenous ribosomal RNA (rRNA) content confirms our data’s authenticity. By performing independent technical library replicates using two high-throughput sequencing platforms, we show not only that aRNA can survive for extended periods in mammalian tissues but also that it has potential for tissue identification. aRNA also has possible further potential, such as identifying in vivo genome activity and adaptation, when sequenced using this technology. [ABSTRACT FROM AUTHOR]

Purpose Recurrence and chemoresistance (CR) are the leading causes of death in patients with high-grade serous carcinoma (HGSC) of the ovary. The aim of this study was to identify genetic changes associated with CR mechanisms using a patient-derived xenograft (PDX) mouse model and genetic sequencing. Materials and Methods To generate a CR HGSC PDX tumor, mice bearing subcutaneously implanted HGSC PDX tumors were treated with paclitaxel and carboplatin. We compared gene expression and mutations between chemosensitive (CS) and CR PDX tumors with whole exome and RNA sequencing and selected candidate genes. Correlations between candidate gene expression and clinicopathological variableswere explored using the Cancer Genome Atlas (TCGA) database and the Human Protein Atlas (THPA). Results Three CR and four CS HGSC PDX tumor models were successfully established. RNA sequencing analysis of the PDX tumors revealed that 146 genes were significantly up-regulated and 54 genes down-regulated in the CR group compared with the CS group. Whole exome sequencing analysis showed 39 mutation sites were identified which only occurred in CR group. Differential expression of SAP25, HLA-DPA1, AKT3, and PIK3R5 genes and mutation of TMEM205 and POLR2A may have important functions in the progression of ovarian cancer chemoresistance. According to TCGA data analysis, patients with high HLADPA1 expression were more resistant to initial chemotherapy (p=0.030; odds ratio, 1.845). Conclusion We successfully established CR ovarian cancer PDX mouse models. PDX-based genetic profiling study could be used to select some candidate genes that could be targeted to overcome chemoresistance of ovarian cancer. [ABSTRACT FROM AUTHOR]

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus. [ABSTRACT FROM AUTHOR]

mTORC1 signaling plays an important role in extracellular and intracellular signals, including growth factors, nutrients, energy metabolism, and stress. However, the functional role of mTORC1 in dentinogenesis is unknown. To study the role of Raptor/mTORC1 in dentinogenesis, an Raptorfl/fl; Osx-Cre (Rap-Osx) mouse, in which Raptor was conditionally deleted in odontoblasts and dental mesenchymal cells, was generated, and postnatal tooth development was compared between Rap-Osx mice and control littermates. Rap-Osx mice presented a phenotype known as dentinogenesis imperfecta and had smaller tooth volume, a thinner dentin layer and a larger pulp chamber. The proliferation and differentiation of odontoblasts/preodontoblasts were attenuated in mutant mice, which was likely responsible for the defects in dentinogenesis. Raptor/mTORC1-pS6K1 signaling was inactivated during tooth development in Rap-Osx mice, whereas it was activated in control mice. These results indicate that Raptor/mTORC1 plays a critical role in dentinogenesis via promoting odontoblasts/preodontoblasts proliferation and differentiation. Raptor/mTORC1 might regulate tooth development through the pS6K1 signaling pathway. [ABSTRACT FROM AUTHOR]

Pruritus (itching) is classically defined as an unpleasant cutaneous sensation that leads to scratching behavior. Although the scientific criteria of classification for pruritic diseases are not clear, it can be divided as acute or chronic by duration of symptoms. In this study, we investigated whether skin injury caused by chemical (contact hypersensitivity, CHS) or physical (skin-scratching stimulation, SSS) stimuli causes initial pruritus and analyzed gene expression profiles systemically to determine how changes in skin gene expression in the affected area are related to itching. In both CHS and SSS, we ranked the Gene Ontology Biological Process terms that are generally associated with changes. The factors associated with upregulation were keratinization, inflammatory response and neutrophil chemotaxis. The Kyoto Encyclopedia of Genes and Genomes pathway shows the difference of immune system, cell growth and death, signaling molecules and interactions, and signal transduction pathways. Il1a, Il1b and Il22 were upregulated in the CHS, and Tnf, Tnfrsf1b, Il1b, Il1r1 and Il6 were upregulated in the SSS. Trpc1 channel genes were observed in representative itching-related candidate genes. By comparing and analyzing RNA-sequencing data obtained from the skin tissue of each animal model in these characteristic stages, it is possible to find useful diagnostic markers for the treatment of itching, to diagnose itching causes and to apply customized treatment. [ABSTRACT FROM AUTHOR]

Dysregulation of sleep and feeding has widespread health consequences. Despite extensive epidemiological evidence for interactions between sleep and metabolic function, little is known about the neural or molecular basis underlying the integration of these processes. D. melanogaster potently suppress sleep in response to starvation, and powerful genetic tools allow for mechanistic investigation of sleep–metabolism interactions. We have previously identified neurons expressing the neuropeptide leucokinin (Lk) as being required for starvation-mediated changes in sleep. Here, we demonstrate an essential role for Lk neuropeptide in metabolic regulation of sleep. The activity of Lk neurons is modulated by feeding, with reduced activity in response to glucose and increased activity under starvation conditions. Both genetic silencing and laser-mediated microablation localize Lk-dependent sleep regulation to a single pair of Lk neurons within the Lateral Horn (LHLK neurons). A targeted screen identified a role for 5′ adenosine monophosphate-activated protein kinase (AMPK) in starvation-modulated changes in sleep. Knockdown of AMPK in Lk neurons suppresses sleep and increases LHLK neuron activity in fed flies, phenocopying the starvation state. Further, we find a requirement for the Lk receptor in the insulin-producing cells (IPCs), suggesting LHLK–IPC connectivity is critical for sleep regulation under starved conditions. Taken together, these findings localize feeding-state–dependent regulation of sleep to a single pair of neurons within the fruit fly brain and provide a system for investigating the cellular basis of sleep–metabolism interactions. [ABSTRACT FROM AUTHOR]

Noroviruses are one of the leading causes of gastro-enteric diseases worldwide in all age groups. Novel epidemic noroviruses with GII.P16 polymerase and GII.2 or GII.4 capsid type have emerged worldwide in late 2015 and in 2016. We performed a molecular epidemiological study of the noroviruses circulating in Italy to investigate the emergence of new norovirus strains. Sentinel hospital-based surveillance, in three different Italian regions, revealed increased prevalence of norovirus infection in children (<15 years) in 2016 (14.4% versus 9.8% in 2015) and the emergence of GII.P16 strains in late 2016, which accounted for 23.0% of norovirus infections. The majority of the strains with a GII.P16 polymerase showed a GII.2 capsid genotype (79.5%). Also, a marked circulation of strains with a GII.17 capsid (14.0%) was observed, chiefly in early 2016. The emergence and global spread of non-GII.4 noroviruses pose challenges for the development of vaccine strategies. [ABSTRACT FROM AUTHOR]

Introduction: Thoracic aortic aneurysm is a potentially fatal disease with a strong genetic contribution. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of this aneurysm. Although previous studies suggested that long non-coding ribonucleic acid (RNA) hypoxia inducible factor 1 α-antisense RNA 1 (HIF1A-AS1) exerted a vital role in the progression and pathogenesis of thoracic aortic aneurysm, we managed to find a new regulatory mechanism of HIF1A-AS1 in VSMCs via transcriptomics., Methods: Cell viability was detected by the cell counting kit-8 assay. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Transwell migration assay and wound healing assay were performed to check the migration ability of HIF1A-AS1 on VSMCs. The NextSeq XTen system (Illumina) was used to collect RNA sequencing data. Lastly, reverse transcription-quantitative polymerase chain reaction confirmed the veracity and reliability of RNA-sequencing results., Results: We observed that overexpressing HIF1A-AS1 successfully promoted apoptosis, significantly altered cell cycle distribution, and greatly attenuated migration in VSMCs, further highlighting the robust promoting effects of HIF1A-AS1 to thoracic aortic aneurysm. Moreover, transcriptomics was implemented to uncover its underlying mechanism. A total of 175 differently expressed genes were identified, with some of them enriched in apoptosis, migration, and cell cycle-related pathways. Intriguingly, some differently expressed genes were noted in vascular development or coagulation function pathways., Conclusion: We suggest that HIF1A-AS1 mediated the progression of thoracic aortic aneurysm by not only regulating the function of VSMCs, but also altering vascular development or coagulation function.

An unusual novel plant virus provisionally named goji berry chlorosis virus (GBCV) was isolated from goji berry plants (Lycium chinense Miller) showing chlorosis symptoms and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 10,100 ribonucleotides and contains six open reading frames (ORFs). Electron microscopy showed that the viral genome is packaged as a filamentous particle with an average length of approximately 850 nm. Phylogenetic analysis and amino acid similarity analysis of the encoded ORFs revealed that this new virus could be classified in an intermediate position between the families Benyviridae and Virgaviridae. The GBCV 200-kDa replicase (ORF1) is more similar to benyvirus replicases than to virgavirus replicases, while its 17-kDa coat protein (CP, ORF2) is more closely related with virgavirus CPs than benyvirus CPs. ORF3 was predicted to produce a C-terminally extended protein from ORF2 via frameshifting. While ORF4 (45-kDa), ORF5 (44-kDa), and ORF6 (16-kDa) have no apparent sequence homology with other known viruses, ORF5 is predicted to encode a movement protein (MP) that is phylogenetically related to the furovirus MP and ORF6 was experimentally proven to encode a viral suppressor of RNA silencing. These unusual characteristics suggest that GBCV may represent an evolutionary link between the families Benyviridae and Virgaviridae and indicate the existence of a novel, unidentified virus group. [ABSTRACT FROM AUTHOR]

Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80–90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection. [ABSTRACT FROM AUTHOR]

Background: A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. Methodology/Principal findings: A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. Conclusions/Significance: The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings. [ABSTRACT FROM AUTHOR]

Cis-regulatory elements (CREs) are one of the important factors in controlling gene expression and elucidation of their roles has been attracting great interest. We have developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our approach, mutant CREs with unique barcode sequences were obtained by biased randomization in a single PCR amplification. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within the range of 0 to 5. The DNA library and subsequent transcribed RNA library were sequenced by next generation sequencers (NGS) to quantify transcriptional activity of each mutant. We succeeded in producing a randomized T7 promoter library with high coverage rate at each target number of base substitutions. In a single NGS analysis, we quantified the transcriptional activity of 7847 T7 promoter variants. We confirmed that the bases from −9 to −7 play an important role in the transcriptional activity of the T7 promoter. This information coincides with the previous researches and demonstrated the validity of our methodology. Furthermore, using an in vitro transcription/translation system, we found that transcriptional activities of these T7 variants were well correlated with the resultant protein abundance. We demonstrate that our method enables simple and high-throughput analysis of the effects of various CRE mutations on transcriptional regulation. [ABSTRACT FROM AUTHOR]

The article discusses research on the role of the phylum Lokiarchaea in the connection between prokaryotes and eukaryotes and in the issue concerning life topology. Topics mentioned include the evidence of contaminated sequences in the Lokiarchaeal genomes, the presence of fast-evolving species, the selection of phylogenetic markers, and the phylogenetic analyses of RNA polymerase.

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations. [ABSTRACT FROM AUTHOR]

SINEUPs are antisense long noncoding RNAs, in which an embedded B2 element -regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest. [ABSTRACT FROM AUTHOR]

A case of melioidosis with a mycotic aneurysm is reported. The blood and tissue isolates were identified as three different species of Burkholderia using the automated identification systems, VITEK 2 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The isolate was confirmed as Burkholderia pseudomallei by 16S rRNA sequencing. The typical features of the Gram staining of colonies and 16S rRNA sequencing can be useful to identify B. pseudomallei.

Cherry virus A (CVA) infection appears to be prevalent in cherry plantations worldwide. In this study, the diversity of CVA isolates from 31 cherry samples collected from different orchards around Bohai Bay in northeastern China was analyzed. The complete genome of one of these isolates, ChYT52, was found to be 7,434 nt in length excluding the poly (A) tail. It shares between 79.9–98.7% identity with CVA genome sequences in GenBank, while its RdRp core is more divergent (79.1–90.7% nt identity), likely as a consequence of a recombination event. Phylogenetic analysis of ChYT52 genome with CVA genomes in Genbank resulted in at least 7 major clusters plus additional 5 isolates alone at the end of long branches suggesting the existence of further phylogroups diversity in CVA. The genetic diversity of Chinese CVA isolates from 31 samples and GenBank sequences were analyzed in three genomic regions that correspond to the coat protein, the RNA-dependent RNA polymerase core region, and the movement protein genes. With few exceptions likely representing further recombination impact, the trees various trees are largely congruent, indicating that each region provides valuable phylogenetic information. In all cases, the majority of the Chinese CVA isolates clustering in phylogroup I, together with the X82547 reference sequence from Germany. Statistically significant negative values were obtained for Tajima’s D in the three genes for phylogroup I, suggesting that it may be undergoing a period of expansion. There was considerable haplotype diversity in the individual samples and more than half samples contained genetically diverse haplotypes belonging to different phylogroups. In addition, a number of statistically significant recombination events were detected in CVA genomes or in the partial genomic sequences indicating an important contribution of recombination to CVA evolution. This work provides a foundation for elucidation of the epidemiological characteristics and evolutionary history of CVA populations. [ABSTRACT FROM AUTHOR]

RNA-seq-based SSU (small subunit) rRNA (ribosomal RNA) analysis has provided a better understanding of potentially active microbial community within environments. However, for RNA-seq library construction, high quantities of purified RNA are typically required. We propose a modified RNA-seq method for SSU rRNA-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10–100 ng) and does not require a DNA removal step. The method was initially tested on three mock communities synthesized with enriched SSU rRNA of archaeal, bacterial and fungal isolates at different ratios, and was subsequently used for environmental samples of high or low biomass. For high-biomass salt-marsh sediments, enriched SSU rRNA and total nucleic acid-derived RNA-seq datasets revealed highly consistent community compositions for all of the SSU rRNA sequences, and as much as 46.4%-59.5% of 16S rRNA sequences were suitable for OTU (operational taxonomic unit)-based community and diversity analyses with complete coverage of V1-V2 regions. OTU-based community structures for the two datasets were also highly consistent with those determined by all of the 16S rRNA reads. For low-biomass samples, total nucleic acid-derived RNA-seq datasets were analyzed, and highly active bacterial taxa were also identified by the OTU-based method, notably including members of the previously underestimated genus Nitrospira and phylum Acidobacteria in tap water, members of the phylum Actinobacteria on a shower curtain, and members of the phylum Cyanobacteria on leaf surfaces. More than half of the bacterial 16S rRNA sequences covered the complete region of primer 8F, and non-coverage rates as high as 38.7% were obtained for phylum-unclassified sequences, providing many opportunities to identify novel bacterial taxa. This modified RNA-seq method will provide a better snapshot of diverse microbial communities, most notably by OTU-based analysis, even communities with low-biomass samples. [ABSTRACT FROM AUTHOR]

Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process. However due to the small number of IRESs known, there have been no systematic investigations of the determinants of IRES activity. With the recent discovery of thousands of novel IRESs in human and viruses, the next challenge is to decipher the sequence determinants of IRES activity. We present the first in-depth computational analysis of a large body of IRESs, exploring RNA sequence features predictive of IRES activity. We identified predictive k-mer features resembling IRES trans-acting factor (ITAF) binding motifs across human and viral IRESs, and found that their effect on expression depends on their sequence, number and position. Our results also suggest that the architecture of retroviral IRESs differs from that of other viruses, presumably due to their exposure to the nuclear environment. Finally, we measured IRES activity of synthetically designed sequences to confirm our prediction of increasing activity as a function of the number of short IRES elements. [ABSTRACT FROM AUTHOR]

Over the last decade, a large number of nucleotide sequences have been generated by next-generation sequencing technologies and deposited to public databases. However, most of these datasets do not specify the sex of individuals sampled because researchers typically ignore or hide this information. Male and female genomes in many species have distinctive sex chromosomes, XX/XY and ZW/ZZ, and expression levels of many sex-related genes differ between the sexes. Herein, we describe how to develop sex marker sequences from syntenic regions of sex chromosomes and use them to quickly identify the sex of individuals being analyzed. Array-based technologies routinely use either known sex markers or the B-allele frequency of X or Z chromosomes to deduce the sex of an individual. The same strategy has been used with whole-exome/genome sequence data; however, all reads must be aligned onto a reference genome to determine the B-allele frequency of the X or Z chromosomes. SEXCMD is a pipeline that can extract sex marker sequences from reference sex chromosomes and rapidly identify the sex of individuals from whole-exome/genome and RNA sequencing after training with a known dataset through a simple machine learning approach. The pipeline counts total numbers of hits from sex-specific marker sequences and identifies the sex of the individuals sampled based on the fact that XX/ZZ samples do not have Y or W chromosome hits. We have successfully validated our pipeline with mammalian (Homo sapiens; XY) and avian (Gallus gallus; ZW) genomes. Typical calculation time when applying SEXCMD to human whole-exome or RNA sequencing datasets is a few minutes, and analyzing human whole-genome datasets takes about 10 minutes. Another important application of SEXCMD is as a quality control measure to avoid mixing samples before bioinformatics analysis. SEXCMD comprises simple Python and R scripts and is freely available at . [ABSTRACT FROM AUTHOR]

PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is one of the main causes of acute viral hepatitis of enteric transmission. HEV has been detected in environmental samples in several countries from Europe and Asia, constituting a risk factor for waterborne infection. In Colombia, HEV has been identified in samples obtained from patients as well as from swine, but no environmental studies have been carried out. To determine if HEV is present in environmental waters, samples from the main source of drinking water plant and of wastewater system of eight municipalities and two villages of Antioquia state (North West Colombia), were collected between December 2012 and April 2014. The HEV genome was detected by RT-PCR in 23.3% (7/30) of the samples from the main source of drinking water plants and in 16.7% (5/30) from sewage. Viral concentrates obtained from three positive sewage samples were used to inoculate HepG2 cell cultures that were followed for one month; however, the viral genome was not detected in any cell culture. This study demonstrates the circulation of HEV in both source of drinking water plants and wastewater in Antioquia state, Colombia. The presence of HEV in environmental waters could be a risk for waterborne transmission in this population. The findings of the present study, together with the evidence of HEV circulation in human and swine in Colombia, should be consider by public health authorities for the development of surveillance programs and the inclusion of HEV infection diagnosis in the guidelines of viral hepatitis in the country. This is the first report of HEV in environmental samples in Colombia and the second one in Latin America. [ABSTRACT FROM AUTHOR]

Introduction: Rates of new HIV-1 diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B HIV-1 subtypes reflecting a growing impact of migration and travel. The present study aims to define HIV-1 subtype diversity patterns and investigate possible HIV-1 transmission networks within Australia. Methods: The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and western Sydney (New South Wales), provided baseline HIV-1 partial pol sequence, age and gender information for 4,873 patients who had genotypes performed during 2005–2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission pairs or clusters (bootstrap ≥98%, genetic distance ≤1.5% from at least one other sequence in the cluster). Results: HIV-1 subtype B represented 74.5% of the 4,873 sequences (WA 59%, SA 68.4%, w-Syd 73.8%, Vic 75.6%, Qld 82.1%), with similar proportion of transmission pairs and clusters found in the B and non-B cohorts (23% vs 24.5% of sequences, p = 0.3). Significantly more subtype B clusters were comprised of ≥3 sequences compared with non-B clusters (45.0% vs 24.0%, p = 0.021) and significantly more subtype B pairs and clusters were male-only (88% compared to 53% CRF01_AE and 17% subtype C clusters). Factors associated with being in a cluster of any size included; being sequenced in a more recent time period (p<0.001), being younger (p<0.001), being male (p = 0.023) and having a B subtype (p = 0.02). Being in a larger cluster (>3) was associated with being sequenced in a more recent time period (p = 0.05) and being male (p = 0.008). Conclusion: This nationwide HIV-1 study of 4,873 patient sequences highlights the increased diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective public health strategies in the current paradigm of HIV prevention in Australia. [ABSTRACT FROM AUTHOR]

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories. [ABSTRACT FROM AUTHOR]

The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection. [ABSTRACT FROM AUTHOR]