High-throughput Sequencing Data Research Articles

Decision Tree Ensembles Utilizing Multivariate Splits Are Effective at Investigating Beta Diversity in Medically Relevant 16S Amplicon Sequencing Data.

Developing an understanding of how microbial communities vary across conditions is an important analytical step. We used 16S rRNA data isolated from human stool samples to investigate whether learned dissimilarities, such as those produced using unsupervised decision tree ensembles, can be used to improve the analysis of the composition of bacterial communities in patients suffering from Crohn's disease and adenomas/colorectal cancers. We also introduce a workflow capable of learning dissimilarities, projecting them into a lower dimensional space, and identifying features that impact the location of samples in the projections. For example, when used with the centered log ratio transformation, our new workflow (TreeOrdination) could identify differences in the microbial communities of Crohn's disease patients and healthy controls. Further investigation of our models elucidated the global impact amplicon sequence variants (ASVs) had on the locations of samples in the projected space and how each ASV impacted individual samples in this space. Furthermore, this approach can be used to integrate patient data easily into the model and results in models that generalize well to unseen data. Models employing multivariate splits can improve the analysis of complex high-throughput sequencing data sets because they are better able to learn about the underlying structure of the data set. IMPORTANCE There is an ever-increasing level of interest in accurately modeling and understanding the roles that commensal organisms play in human health and disease. We show that learned representations can be used to create informative ordinations. We also demonstrate that the application of modern model introspection algorithms can be used to investigate and quantify the impacts of taxa in these ordinations, and that the taxa identified by these approaches have been associated with immune-mediated inflammatory diseases and colorectal cancer.

Enhanced external counterpulsation improves dysfunction of forearm muscle caused by radial artery occlusion.

This study aimed to investigate the therapeutic effect of enhanced external counterpulsation (EECP) on radial artery occlusion (RAO) through the oscillatory shear (OS) and pulsatile shear (PS) models of human umbilical vein endothelial cells (HUVECs) and RAO dog models. We used high-throughput sequencing data GSE92506 in GEO database to conduct time-series analysis of functional molecules on OS intervened HUVECs, and then compared the different molecules and their functions between PS and OS. Additionally, we studied the effect of EECP on the radial artery hemodynamics in Labrador dogs through multi-channel physiological monitor. Finally, we studied the therapeutic effect of EECP on RAO at the histological level through Hematoxylin-Eosin staining, Masson staining, ATPase staining and immunofluorescence in nine Labrador dogs. With the extension of OS intervention, the cell cycle decreased, blood vessel endothelial cell proliferation and angiogenesis responses of HUVECs were down-regulated. By contrast, the inflammation and oxidative stress responses and the related pathways of anaerobic metabolism of HUVECs were up-regulated. Additionally, we found that compared with OS, PS can significantly up-regulate muscle synthesis, angiogenesis, and NO production related molecules. Meanwhile, PS can significantly down-regulate inflammation and oxidative stress related molecules. The invasive arterial pressure monitoring showed that 30Kpa EECP treatment could significantly increase the radial artery peak pressure (p = 0.030, 95%CI, 7.236-82.524). Masson staining showed that RAO significantly increased muscle interstitial fibrosis (p = 0.002, 95%CI, 0.748-2.128), and EECP treatment can reduce this change (p = 0.011, 95%CI, -1.676 to -0.296). ATPase staining showed that RAO significantly increased the area of type II muscle fibers (p = 0.004, 95%CI, 7.181-25.326), and EECP treatment could reduce this change (p = 0.001, 95%CI, -29.213 to -11.069). In addition, immunofluorescence showed that EECP increased angiogenesis in muscle tissue (p = 0.035, 95%CI, 0.024-0.528). EECP improves interstitial fibrosis and hypoxia, and increases angiogenesis of muscle tissue around radial artery induced by RAO.

Abstract P5-02-41: UBE2E3 promotes the progression of HER2-positive breast cancer and influences the efficacy of targeted therapy via EGFR stabilization

Abstract Background: In the past 20 years, the efficacy and prognosis of HER2-positive breast cancer have significantly improved. However, nearly 50% of patients still have residual invasive tumors after chemotherapy combined with dual-targeted neoadjuvant therapy, especially for those with disease progression during treatment. A lack of effective therapeutic regimens results from the failure of targeted therapy, whose heterogeneity is especially worthy of our attention. The aim of this study was to look for efficacy markers and investigate new drug-resistance mechanisms. Methods: Firstly, the high-throughput sequencing data from 81 patients who received neoadjuvant chemotherapy TCbH (paclitaxel + carboplatin + trastuzumab) was analyzed by the efficacy outcomes. They were divided into 8 patients with stable or progressive disease (SD/PD), 35 with partial response (PR), and 38 with pathological complete remission (pCR). Then, UBE2E3 was chosen from the different expression genes between SD/PD and pCR based on efficacy results and the weighted gene co-expression network (WGCNA). UBE2E3 clinical correlations were investigated using publicly available data from The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and UBE2E3 was validated using immunohistochemistry (IHC) on 200 HER2-positive breast cancer tissue chips. Further, the UBE2E3 knockdown and overexpression stable transfer cell lines were constructed, and the effects of UBE2E3 on cell proliferation, clone formation, and drug sensitivity were verified by live cell imaging, the CCK8 assay, plate cloning, and IC50 assays, respectively. The tumor growth of UBE2E3 in vivo was investigated by an in situ transplantation tumor assay in nude mice. Meanwhile, the p-RB assay of mouse tissues by IHC was used to explore the effect of UBE2E3 on cell proliferation. RNA-seq was used to screen the downstream molecules of UBE2E3. Western blotting was used to verify the results of bioinformatics analysis and to explore the downstream key molecules. The protease inhibitor MG132 and actinomycin CHX were used to look at the effect on the stability of the target protein. Immunoprecipitation and silver staining assays were used to find interacting proteins with the UBE2E3. Results: Ten hub-genes which were efficacy-related were identified by WGCNA analysis, in which UBE2E3 was highly expressed in the SD/PD group (p < 0.05). In HER2-positive breast cancer, high expression of UBE2E3 was associated with poor prognosis and decreased disease-free survival both in public data and Fudan University Shanghai Cancer Center (FUSCC) data [HR 2. 36, (1.25–4.47), p < 0.05]. The experimental results demonstrated that UBE2E3 promoted the proliferation of HER2-positive breast cancer cells, enhanced clone formation, and resisted lapatinib’s treatment in cellular phenotype; and that UBE2E3 promoted tumor growth in vivo and upregulated the expression of p-RB. The differentially expressed genes’ sets of the RNA-seq between overexpressed cell lines and control showed that overexpressing UBE2E3 activated the EGFR pathway. Further, an immunoblot assay confirmed that UBE2E3 positively regulated EGFR levels and activated the downstream MAPK pathway. The proteasome inhibitor MG132 and CHX assays showed that UBE2E3 could stabilize EGFR proteins. The co-immunoprecipitation and silver staining assays showed that UBE2E3 stabilized EGFR proteins by interacting with c-Cbl. Conclusion: UBE2E3 could negatively affect the efficacy of HER2-positive breast cancer therapy and is significantly associated with poor prognosis. UBE2E3 may serve as a potential marker of efficacy and prognosis for HER2-positive breast cancer in the future. Therapeutic efficacy is affected by UBE2E3, which binds to c-Cbl and causes upregulation of EGFR expression in vivo, which in turn causes the MAPK pathway to be activated and tumor growth to be pushed up. Citation Format: Pei Li, Wei-Ru Chi, Bingqiu Xiu, Qi Zhang, Liyi Zhang, Ming Chen, Jingyan Xue, Xiaoyan Huang, Yayun Chi, Jiong Wu. UBE2E3 promotes the progression of HER2-positive breast cancer and influences the efficacy of targeted therapy via EGFR stabilization [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-41.

Bioinformatics analysis based on high-throughput sequencing data to identify hub genes related to different clinical types of COVID-19.

This article aims to explore hub genes related to different clinical types of cases with COVID-19 and predict the therapeutic drugs related to severe cases. The expression profile of GSE166424 was divided into four data sets according to different clinical types of COVID-19 and then calculated the differential expression genes (DEGs). The specific genes of four clinical types of COVID-19 were obtained by Venn diagram and conducted enrichment analysis, protein-protein interaction (PPI) networks analysis, screening hub genes, and ROC curve analysis. The hub genes related to severe cases were verified in GSE171110, their RNA-specific expression tissues were obtained from the HPA database, and potential therapeutic drugs were predicted through the DGIdb database. There were 536, 266, 944, and 506 specific genes related to asymptomatic infections, mild, moderate, and severe cases, respectively. The hub genes of severe specific genes were AURKB, BRCA1, BUB1, CCNB1, CCNB2, CDC20, CDC6, KIF11, TOP2A, UBE2C, and RPL11, and also differentially expressed in GSE171110 (P < 0.05), and their AUC values were greater than 0.955. The RNA tissue specificity of AURKB, CDC6, KIF11, UBE2C, CCNB2, CDC20, TOP2A, BUB1, and CCNB1 specifically enhanced on lymphoid tissue; CCNB2, CDC20, TOP2A, and BUB1 specifically expressed on the testis. Finally, 55 drugs related to severe COVID-19 were obtained from the DGIdb database. Summary, AURKB, BRCA1, BUB1, CCNB1, CCNB2, CDC20, CDC6, KIF11, TOP2A, UBE2C, and RPL11 may be potential diagnostic biomarkers for severe COVID-19, which may affect immune and male reproductive systems. 55 drugs may be potential therapeutic drugs for severe COVID-19.

An Integrated Pipeline for Trio-Rapid Genome Sequencing in Critically Ill Infants.

Trio-rapid genome sequencing (trio-rGS) can assist the genetic diagnosis of critically ill infants given its ability to detect a broad range of pathogenic variants, as well as microbes, simultaneously with high efficiency. To achieve more comprehensive clinical diagnoses, it is essential to propose a recommended protocol in clinical practice. Here, we introduced an integrated pipeline to detect germline variants and microorganisms simultaneously from trio-RGS in critically ill infants, which provides step-by-step criteria for the semi-automatic processing procedures. With this pipeline in clinical application, only 1 ml of peripheral blood is needed for clinicians to provide both genetic and infectious causal information to a patient. The establishment and clinical practice of the method is of great significance for further mining of high-throughput sequencing data and for assisting clinicians in promoting diagnosis efficiency and accuracy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Experimental pipeline for rapid whole-genome sequencing for the simultaneous detection of germline variants and microorganisms Basic Protocol 2: Computational pipeline for rapid whole-genome sequencing for the simultaneous detection of germline variants and microorganisms.

First report of Watermelon crinkle leaf-associated virus 2 infecting watermelon (Citrullus lanatus) in Oklahoma.

Watermelon (Citrullus lanatus) is one of the major cucurbit crops in Oklahoma, which is grown on >1,200 acres. It contributes $16 million to the state's economy (Shrefler et al., 2017; NASS, 2022). During the 2021 growing season, virus-like symptoms on watermelon plants were observed in a grower's field in Blaine County, Oklahoma. These symptoms included mottling, crinkled leaf edges, and a unique yellow mosaic pattern (Fig. 1). Leaf tissue samples were randomly collected from nine plants and total RNA was extracted using Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNA from one sample (named BL13) was subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 High-Output kit v2.5 (Illumina, USA) at the genomic facility of Oklahoma State University. A total of 21,52,786 trimmed pair-end reads were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc.) and subjected to BLASTn analysis. Of these, three contigs of 6,626 bp, 1,383 bp and 1,233 bp (average coverage 20,658X) showed 99% nucleotide (nt) sequence identity with RNA1 of a watermelon crinkle leaf-associated virus (WCLaV-2) isolate from Brazil (LC636073). The HTS data for BL13 samples also revealed three contigs of 263 pb, 567 bp and 1,066 bp that matched with cucumis melo crytpic virus isolate CmCV-HLI but was not confirmed by RT-PCR. The presence of WCLaV-2 in the BL13 sample was confirmed by RT-PCR using primers (RNA1-F: 5'-CAAAACGCCCATAAG AATAG-3'and RNA1-R: 5'- CTCCATTAATGAGAACTAGGAG-3') which amplified a 1.2 kb segment of the RNA1. The PCR product was confirmed on 1% agarose gel, cloned and three independent recombinant clones Sanger sequenced (Eurofins Genomics LLC, Louisville, KY, USA). The resulting consensus sequence (1.2 Kb) (ON453838) was subjected to a BLASTn search against the Genbank databases, resulting in 99.76% nt identity with WCLaV-2 RNA1 (Accession no. LC636073). Screening of the remaining eight symptomatic samples by RT-PCR revealed another positive sample (named BL7) for WCLaV-2. Consensus nt sequence (1.2 Kb) analysis for the virus isolate from the BL7 sample (Accession no. ON453839) shared 99.84% nt identities with WCLaV-2 RNA1 (LC636073). Total RNA from BL13 and BL7 samples, as well as the remaining seven samples, were tested against three major potyviruses (PRSV-W, WMV, and ZYMV) by multiplex RT-PCR (Rajbanshi & Ali, 2019). Both BL13 and BL7 samples were negative to all three potyviruses but the other eight samples were positive to one or two of the potyviruses. WCLaV-2 is a negative-sense ssRNA virus (family Phenuiviridae) and has been recently reported from watermelon in China (Xin et al., 2017) and Brazil (Maeda et al. 2022). In the United States, it has been reported from Florida (Hendrick et al., 2021), Georgia (Adeleke et al., 2022), and Texas (Hernandez et al., 2021). The mode of transmission of WCLaV-2 and its direct effect on watermelon fruit is not known (Xin et al., 2017; Maeda et al., 2022). Our results report for the first time the occurrence of WCLaV-2 infecting watermelon naturally in Oklahoma and show a further potential threat to watermelon production in the State. Further studies are needed to determine the incidence of WCLaV-2 in other counties of Oklahoma.

Reconstructing B cell lineage trees with minimum spanning tree and genotype abundances

B cell receptor (BCR) genes exposed to an antigen undergo somatic hypermutations and Darwinian antigen selection, generating a large BCR-antibody diversity. This process, known as B cell affinity maturation, increases antibody affinity, forming a specific B cell lineage that includes the unmutated ancestor and mutated variants. In a B cell lineage, cells with a higher antigen affinity will undergo clonal expansion, while those with a lower affinity will not proliferate and probably be eliminated. Therefore, cellular (genotype) abundance provides a valuable perspective on the ongoing evolutionary process. Phylogenetic tree inference is often used to reconstruct B cell lineage trees and represents the evolutionary dynamic of BCR affinity maturation. However, such methods should process B-cell population data derived from experimental sampling that might contain different cellular abundances. There are a few phylogenetic methods for tracing the evolutionary events occurring in B cell lineages; best-performing solutions are time-demanding and restricted to analysing a reduced number of sequences, while time-efficient methods do not consider cellular abundances. We propose ClonalTree, a low-complexity and accurate approach to construct B-cell lineage trees that incorporates genotype abundances into minimum spanning tree (MST) algorithms. Using both simulated and experimental data, we demonstrate that ClonalTree outperforms MST-based algorithms and achieves a comparable performance to a method that explores tree-generating space exhaustively. Furthermore, ClonalTree has a lower running time, being more convenient for building B-cell lineage trees from high-throughput BCR sequencing data, mainly in biomedical applications, where a lower computational time is appreciable. It is hundreds to thousands of times faster than exhaustive approaches, enabling the analysis of a large set of sequences within minutes or seconds and without loss of accuracy. The source code is freely available at github.com/julibinho/ClonalTree.

First Report of Tomato Yellow Leaf Curl Virus Infecting Pepper and Tomato in Oklahoma.

Tomato yellow leaf curl virus (TYLCV), (genus Begomovirus and family Geminiviridae) is one of the devastating viruses infecting tomatoes (Solanum Lycopersicon) and could have a significant impact on tomato production worldwide (Moriones and Navas-Castillo, 2000; Rybicki, 2015; Perez-Padilla et al. 2019). TYLCV was first reported in the United States (US) in 1997 in Florida (Polston et al. 1999) and since then it has been reported in several other states but not in Oklahoma (de Sa et al. 2008). Both pepper (Capsicum spp.) and tomato are grown in >50 counties of Oklahoma for fresh produce in the local market, and for processing industries. During a survey in 2021, pepper (bell pepper cv SV3964) and tomato (cv grand marshall) plants grown in a commercial field in Bixby, Oklahoma, showed typical virus-like symptoms including yellowing, leaf curling, cupping, twisting, and mottling (Fig. 1). Estimated disease incidence was 10% in both crops. In addition, whiteflies (Bemisia tabaci) were also observed on these plants. Twenty symptomatic samples from pepper (n=16) and tomato (n=4) plants, and four asymptomatic from pepper (n=2) and tomato (n=2) plants were collected and brought to the Plant Virology Lab at the University of Tulsa for further analysis Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, LOT: SLCG7913). RNA from two symptomatic samples, one each from pepper and tomato (named BX6 and BX11, respectively) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 22,385,404 (average length 73.6 bp) and 19,012,605 (average length 73.7 bp) trim pair-end for both samples (BX6 and BX11) were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. The three contigs: 393 bp (coverage 363.24X), 670 bp (coverage 489.92X), and 990 bp (coverage 112.96X) for BX6 isolate, and four contigs: 393 bp (coverage 341.64X), 668 bp (coverage 457.23X), 814 bp (coverage 107.86X), and 990 bp (coverage 93.71X), for BX11 isolate, showed from 96 to 99% nucleotide (nt) identities with movement protein, capsid protein, C3, V2, C4, AC1, AC2, AC3 protein genes of the TYLCV isolates from Australia, China, Iran, Mexico, South Korea, and the US. The HTS data did not reveal any other viral sequence in these two samples. To further confirm the presence of TYLCV in these samples, three overlapping TYLCV-specific primer pairs (Supplementary Table 1) were designed based on the alignment of complete genome sequences of TYLCV isolates present in the Genbank. Total DNA was extracted from fresh leaf tissues of BX6 and BX11 samples using the Plant DNA Kit (Omega BIO-TEK,) and used in the PCR assay. The expected PCR products (814 bp, 1,085 bp and 1,068 bp) were generated from both samples providing further evidence of TYLCV infection. To obtain the complete genome sequence, these PCR amplified DNA fragments of both isolates were gel extracted and cloned using the pGEM-T® Easy Vector system. Three independent clones of each fragment were sequenced and analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the GenBank and had the highest identities (99-99.2%) with TYLCV sequences. The overlapping consensus nt sequence of the BX6 isolate was 2,782 nt (Accession no ON321843), whereas that of BX11 was 2,777 nt (Accession no ON785706) showed 99.2% and 99.0% nt identities with the corresponding sequence of TYLCV isolates (KX347141 and KX347142) respectively from Australia. The nt identity between BX6 and BX11 was 99.7%. Further screening by PCR of the remaining 18 symptomatic field samples (pepper, n=15 and tomatoes, n=3) revealed TYLCV infection in two pepper and all three tomato samples consistent with the greater susceptibility of tomato. None of the asymptomatic pepper and tomato samples were positive for TYLCV. DNA from both BX6 and BX11 samples was also used in the rolling circle amplification (RCA) assay (TempliPhi Amplification Kit, Cytiva) and the presence of circular TYLCV DNA was detected. Our results demonstrated the presence of TYLCV infection in these symptomatic pepper and tomato plants. This is the first report of TYLCV infecting pepper or tomato in open fields in Oklahoma. Further surveys are needed to determine the incidence of TYLCV and the presence of its vector in other counties throughout Oklahoma.

PFresGO: an attention mechanism-based deep-learning approach for protein annotation by integrating gene ontology inter-relationships.

The rapid accumulation of high-throughput sequence data demands the development of effective and efficient data-driven computational methods to functionally annotate proteins. However, most current approaches used for functional annotation simply focus on the use of protein-level information but ignore inter-relationships among annotations. Here, we established PFresGO, an attention-based deep-learning approach that incorporates hierarchical structures in Gene Ontology (GO) graphs and advances in natural language processing algorithms for the functional annotation of proteins. PFresGO employs a self-attention operation to capture the inter-relationships of GO terms, updates its embedding accordingly and uses a cross-attention operation to project protein representations and GO embedding into a common latent space to identify global protein sequence patterns and local functional residues. We demonstrate that PFresGO consistently achieves superior performance across GO categories when compared with 'state-of-the-art' methods. Importantly, we show that PFresGO can identify functionally important residues in protein sequences by assessing the distribution of attention weightings. PFresGO should serve as an effective tool for the accurate functional annotation of proteins and functional domains within proteins. PFresGO is available for academic purposes at https://github.com/BioColLab/PFresGO. Supplementary data are available at Bioinformatics online.

Assembly-free quantification of vagrant DNA inserts.

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called "vagrantDNA" and code to simulate test data sets.

Globally nitrogen addition alters soil microbial community structure, but has minor effects on soil microbial diversity and richness

Globally increasing nitrogen (N) deposition is recognized as an important regulator of soil microbial communities. However, how N enrichment affects soil microbial diversity, richness and community structure remains unclear at the global scale. Here, by focusing on high-throughput amplicon sequencing data from field experiments using N fertilizers only, we conducted a meta-analysis of a global dataset assessing the responses of microbial diversity (Shannon index), richness (Chao1, OTU richness) and community structure to N addition. Our results showed that N addition significantly reduced soil bacterial diversity (−2.3%), and such effect was significant in cropland (rather than grassland and forest) and with urea addition (rather than ammonium nitrate). However, there was no significant effect of N addition on soil fungal diversity and microbial (bacterial or fungal) richness. Moreover, N addition shifted microbial community structure likely due to the microbial adaptation to N-excess, but had no significant effect on microbial beta-diversity. Model-selection analysis further showed that the change in soil pH was the most important factor regulating the responses of soil bacterial diversity and richness to N addition. Overall, our results contribute to an in-depth understanding of the effects of N addition on soil microbial diversity and community structure in terrestrial ecosystems at the global scale.

The phylogeny and global biogeography of Primulaceae based on high-throughput DNA sequence data

The angiosperm family Primulaceae is morphologically diverse and distributed nearly worldwide. However, phylogenetic uncertainty has obstructed the identification of major morphological and biogeographic transitions within the clade. We used target capture sequencing with the Angiosperms353 probes, taxon-sampling encompassing nearly all genera of the family, tree-based sequence curation, and multiple phylogenetic approaches to investigate the major clades of Primulaceae and their relationship to other Ericales. We generated dated phylogenetic trees and conducted broad-scale biogeographic analyses as well as stochastic character mapping of growth habit. We show that Ardisia, a pantropical genus and the largest in the family, is not monophyletic, with at least 19 smaller genera nested within it. Neotropical members of Ardisia and several smaller genera form a clade, an ancestor of which arrived in the Neotropics and began diversifying about 20 Ma. This Neotropical clade is most closely related to Elingamita and Tapeinosperma, which are most diverse on islands of the Pacific. Both Androsace and Primula are non-monophyletic by the inclusion of smaller genera. Ancestral state reconstructions revealed that there have either been parallel transitions to an herbaceous habit in Primuloideae, Samolus, and at least three lineages of Myrsinoideae, or a common ancestor of nearly all Primulaceae was herbaceous. Our results provide a robust estimate of phylogenetic relationships across Primulaceae and show that a revised classification of Myrsinoideae and several other clades within the family is necessary to render all genera monophyletic.

DraculR: A Web-Based Application for In Silico Haemolysis Detection in High-Throughput microRNA Sequencing Data.

The search for novel microRNA (miRNA) biomarkers in plasma is hampered by haemolysis, the lysis and subsequent release of red blood cell contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multicompartment origin and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to sample. The inclusion of red-blood-cell-derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify posthoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload miRNA expression data from a short-read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. The code, DraculR web tool and its tutorial are freely available as detailed herein.

Xenbase: key features and resources of the Xenopus model organism knowledgebase.

Xenbase (https://www.xenbase.org/), the Xenopus model organism knowledgebase, is a web-accessible resource that integrates the diverse genomic and biological data from research on the laboratory frogs Xenopus laevis and Xenopus tropicalis. The goal of Xenbase is to accelerate discovery and empower Xenopus research, to enhance the impact of Xenopus research data, and to facilitate the dissemination of these data. Xenbase also enhances the value of Xenopus data through high-quality curation, data integration, providing bioinformatics tools optimized for Xenopus experiments, and linking Xenopus data to human data, and other model organisms. Xenbase also plays an indispensable role in making Xenopus data interoperable and accessible to the broader biomedical community in accordance with FAIR principles. Xenbase provides annotated data updates to organizations such as NCBI, UniProtKB, Ensembl, the Gene Ontology consortium, and most recently, the Alliance of Genomic Resources, a common clearing house for data from humans and model organisms. This article provides a brief overview of key and recently added features of Xenbase. New features include processing of Xenopus high-throughput sequencing data from the NCBI Gene Expression Omnibus; curation of anatomical, physiological, and expression phenotypes with the newly created Xenopus Phenotype Ontology; Xenopus Gene Ontology annotations; new anatomical drawings of the Normal Table of Xenopus development; and integration of the latest Xenopus laevis v10.1 genome annotations. Finally, we highlight areas for future development at Xenbase as we continue to support the Xenopus research community.

Investigating the molecular mechanisms of delirium-like neuropsychiatric disorder induced by electromagnetic pulse based on bioinformatics analysis

Electromagnetic pulse (EMP), a unique type of electromagnetic radiation, may induce diverse neuropsychiatric disorders, such as irritability, hyperkinesis, retardation of learning and memory. However, the underlying mechanism of EMP exposure on neuronal injury has not been elucidated. Here, we aimed to delineate the regulatory expression networks based on high-throughput sequencing data to explore the possible molecular mechanisms related to EMP-induced delirium-like neuropsychiatric disorder in rats. It’s shown that EMP exposure induced anxiety, cognitive decline and short-term memory impairment. The expression profiles of the long noncoding RNAs (lncRNAs) and mRNAs, along with their biological function and regulatory network, were explored in rats after EMP exposure. We identified 41 differentially expressed lncRNAs (DELs) and 266 differentially expressed mRNAs (DEMs) between EMP and sham groups. Sixty-one co-expression relationships between 18 DELs and 56 DEMs were mostly associated with synapse- and metabolic-related pathways. We predicted 51 DEL-miRNA pairs and 290 miRNA-mRNA pairs using the miRanda database to constructed a DEL-miRNA-DEM network. LncRNA AABR07042999.1 and mRNA Tph2, Slc6a4, Dbh and Th were upregulated, and the contents of serotonin, dopamine and norepinephrine were increased in both PFC and HIP after EMP exposure. The current study provided a better understanding of the ceRNA network, which might reveal the pathological mechanism and provide more treatment options for the EMP-induced neurobehavioral disorder.

VHL-HIF-2α axis-induced SEMA6A upregulation stabilized β-catenin to drive clear cell renal cell carcinoma progression

SEMA6A is a multifunctional transmembrane semaphorin protein that participates in various cellular processes, including axon guidance, cell migration, and cancer progression. However, the role of SEMA6A in clear cell renal cell carcinoma (ccRCC) is unclear. Based on high-throughput sequencing data, here we report that SEMA6A is a novel target gene of the VHL-HIF-2α axis and overexpressed in ccRCC. Chromatin immunoprecipitation and reporter assays revealed that HIF-2α directly activated SEMA6A transcription in hypoxic ccRCC cells. Wnt/β-catenin pathway activation is correlated with the expression of SEMA6A in ccRCC; the latter physically interacted with SEC62 and promoted ccRCC progression through SEC62-dependent β-catenin stabilization and activation. Depletion of SEMA6A impaired HIF-2α-induced Wnt/β-catenin pathway activation and led to defective ccRCC cell proliferation both in vitro and in vivo. SEMA6A overexpression promoted the malignant phenotypes of ccRCC, which was reversed by SEC62 depletion. Collectively, this study revealed a potential role for VHL-HIF-2α-SEMA6A-SEC62 axis in the activation of Wnt/β-catenin pathway. Thus, SEMA6A may act as a potential therapeutic target, especially in VHL-deficient ccRCC.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Prevent Acute Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation Via Exosome-Mediated MicroRNA and Its Mechanism

To explore molecular mechanisms by which umbilical cord-derived mesenchymal stem cells suppress the development of GVHD after bone marrow hematopoietic stem cell transplantation. A mouse model of aGVHD was constructed after bone marrow hematopoietic stem cell transplantation, and the umbilical cord-derived mesenchymal stem cells were cultured, and then injected into the aGVHD mouse model, so as to investigate its prophylactic efficacy. Prophylactic effect of the exosomes isolated from umbilical cord-derived mesenchymal stem cells on aGVHD mice was assessed. Sequencing analysis of miRNA from exosomes was performed. aGVHD model was successfully constructed after hematopoietic stem cell transplantation. By injecting umbilical cord-derived mesenchymal stem cells into the GVHD mouse model, it was found that the treatment significantly prolonged survival time of mice compared to the untreated group. Injection exosomes derived from umbilical cord-derived mesenchymal stem cells into the GVHD mouse model significantly prolonged the survival time of mice compared to the untreated group. High-throughput sequencing data showed that microRNA such as miR-21 in exosomes isolated from umbilical cord-derived mesenchymal stem cells, which mainly affected the signaling pathways such as cell adhesion, RNA degradation. The umbilical cord-derived mesenchymal stem cells can prevent the occurrence of aGVHD after HSCT, which is mediate by MicroRNA in the exosomes derived from umbilical cord-derived mesenchymal stem cells.

Transcriptomic Analysis of Flowering Time Genes in Cultivated Chickpea and Wild Cicer.

Chickpea (Cicer arietinum L.) is a major grain legume and a good source of plant-based protein. However, comprehensive knowledge of flowering time control in Cicer is lacking. In this study, we acquire high-throughput transcriptome sequencing data and analyze changes in gene expression during floral transition in the early flowering cultivar ICCV 96029, later flowering C. arietinum accessions, and two wild species, C. reticulatum and C. echinospermum. We identify Cicer orthologs of A. thaliana flowering time genes and analyze differential expression of 278 genes between four species/accessions, three tissue types, and two conditions. Our results show that the differences in gene expression between ICCV 96029 and other cultivated chickpea accessions are vernalization-dependent. In addition, we highlight the role of FTa3, an ortholog of FLOWERING LOCUS T in Arabidopsis, in the vernalization response of cultivated chickpea. A common set of differentially expressed genes was found for all comparisons between wild species and cultivars. The direction of expression change for different copies of the FT-INTERACTING PROTEIN 1 gene was variable in different comparisons, which suggests complex mechanisms of FT protein transport. Our study makes a contribution to the understanding of flowering time control in Cicer, and can provide genetic strategies to further improve this important agronomic trait.

Detecting genomic deletions from high-throughput sequence data with unsupervised learning

BackgroundStructural variation (SV), which ranges from 50 bp to sim 3 Mb in size, is an important type of genetic variations. Deletion is a type of SV in which a part of a chromosome or a sequence of DNA is lost during DNA replication. Three types of signals, including discordant read-pairs, reads depth and split reads, are commonly used for SV detection from high-throughput sequence data. Many tools have been developed for detecting SVs by using one or multiple of these signals.ResultsIn this paper, we develop a new method called EigenDel for detecting the germline submicroscopic genomic deletions. EigenDel first takes advantage of discordant read-pairs and clipped reads to get initial deletion candidates, and then it clusters similar candidates by using unsupervised learning methods. After that, EigenDel uses a carefully designed approach for calling true deletions from each cluster. We conduct various experiments to evaluate the performance of EigenDel on low coverage sequence data.ConclusionsOur results show that EigenDel outperforms other major methods in terms of improving capability of balancing accuracy and sensitivity as well as reducing bias. EigenDel can be downloaded from https://github.com/lxwgcool/EigenDel.

Knockdown of pyruvate kinase M2 suppresses bladder cancer progression

Abstract Objectives Bladder cancer (BCa) is one of the most frequently diagnosed cancers of the urinary tract and has a high mortality. The M2 splice isoform of pyruvate kinase (PKM2) is a key regulator of the Warburg effect in cancer cells. This study aimed to evaluate metabolic alterations and biological behaviours after knocking down PKM2. Methods In this study, 36 pairs of BCa tissues and adjacent normal tissues were collected to analyse the expression level of PKM2 and to explore the relationship between PKM2 level and tumour and patient status. After PKM2 knockdown in T24 cells, cell survival, migration, invasion, glucose uptake, lactate production, and apoptosis were detected. The tumour-forming ability of PKM2-reducing T24 cells was examined in vivo. Results The results showed that PKM2 expression correlates with BCa stage and grade. PKM2 knockdown decreases glucose consumption and lactate production and suppresses cell proliferation, migration, and invasion while increasing reactive oxygen species levels and apoptosis in T24 BCa cells in vitro. In nude mouse models, PKM2 knockdown reduced xenograft and orthotopic tumour size. Moreover, PKM2 knockdown decreased vimentin and fibronectin expression and increased E-cadherin expression. Analysis of high-throughput sequencing data revealed that PKM2 may also be associated with biological processes and diseases. Conclusions Overall, these results indicate that PKM2 may be a therapeutic target for BCa patients.