Analysis Of High-throughput Sequencing Data Research Articles

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Diversity of fungal communities on Cabernet and Aglianico grapes from vineyards located in Southern Italy.

Grape-associated microbial community is influenced by a combination of viticultural, climatic, pedological and anthropological factors, collectively known as terroir. Therefore, grapes of the same cultivar grown in different areas can be appreciated for their distinctive biogeographic characteristics. In our previous study, we showed that the phenotypic response of Aglianico and Cabernet grapevines from Molise and Sicily regions is significantly influenced by the prevailing pedoclimatic conditions, particularly soil physical properties. However, the scale at which microbial communities differ could be important in clarifying the concept of terroir, including whether it is linked to the grape variety present in a particular vineyard. To explore this further, in the research presented here, a comparative study on the fungal communities inhabiting the berry surfaces of Cabernet and Aglianico cultivars was conducted on different vineyards located in Southern Italy (Molise, Sicily and Campania regions, the first two of which had been involved in our previous study) by using high-throughput sequencing (HTS) and multivariate data analysis. The descriptive approach through relative abundance analysis showed the most abundant phyla (Ascomycota, Basidiomycota, and Chytridiomycota), families (Cladosporiaceae, Saccotheciaceae, Pleosporaceae, Saccharomycodaceae, Sporidiobolaceae, Didymellaceae, Filobasidiaceae, Bulleribasidiaceae, and Saccharomycetaceae) and genera (Cladosporium, Aureobasidium, Alternaria, Stemphylium and Filobasidium) detected on grape berries. The multivariate data analysis performed by using different packages (phyloseq, Vegan, mixOmics, microbiomeMarker and ggplot2) highlighted that the variable "vineyard location" significantly affect the fungal community, while the variable "grape variety" has no significant effect. Thus, some taxa are found to be part of specific vineyard ecosystems rather than specific grape varieties, giving additional information on the microbial contribution to wine quality, thanks to the presence of fermentative yeasts or, conversely, to the involvement in negative or detrimental roles, due to the presence of grape-deriving fungi implied in the spoilage of wine or in grapevine pathogenesis. In this connection, the main functions of core taxa fungi, whose role in the vineyard environment is still poorly understood, are also described.

Human Breast Milk Exosomal miRNAs are Influenced by Premature Delivery and Affect Neurodevelopment.

This study investigates the exosomal microRNA (miRNA) profiles of term and preterm breast milk, including the most abundant and differentially expressed (DE) miRNAs, and their impact on neurodevelopment in infants. Mature milk is collected from the mothers of term and preterm infants. Using high-throughput sequencing and subsequent data analysis, exosomal miRNA profiles of term and preterm human breast milk (HBM) are acquired and it is found that the let-7 and miR-148 families are the most abundant miRNAs. Additionally, 23 upregulated and 15 downregulated miRNAs are identified. MiR-3168 is the most upregulated miRNA in preterm HBM exosome, exhibiting targeting activity toward multiple genes involved in the SMAD and MAPK signaling pathways and playing a crucial role in early neurodevelopment. Additionally, the effects of miR-3168 on neurodevelopment is confirmed and it is determined that it is an essential factor in the differentiation of neural stem cells (NSCs). This study demonstrates that miRNA expression in breast milk exosomes can be influenced by preterm delivery, thereby potentially impacting neurodevelopment in preterm infants.

Microbial community structures and bacteria-Cylindrospermopsis raciborskii interactions in Yilong Lake.

Cylindrospermopsis raciborskii-dominated harmful algae blooms have been reported globally in recent years. However, our understanding of the ecology of C. raciborskii in natural conditions is still poor. In this study, we collected the water samples from a C. raciborskii-blooming lake, Yilong Lake, in Yunnan province, China, and used both culture-dependent and culture-independent approaches to investigate their microbial communities and the interactions between C. raciborskii and the other bacteria. The composition and diversity of microbial communities were revealed with 16S rRNA gene high-throughput sequencing data analysis. Microbial co-occurrences analysis suggests C. raciborskii may have complex associations with other bacteria. Based on co-inoculation tests, we obtained 14 strains of bacterial strains from the water samples that exhibited either algicidal or promoting effects on a strain of C. raciborskii. Two bacterial isolates exhibited a consistent performance between co-occurrence analysis and experimental results. Effects of these bacteria-algae interspecies interactions on the bloom event are discussed. All these results may provide new insights into the C. raciborskii-dominated blooms and how its interspecies relationships with other bacteria may influence the bloom events in eutrophic waters throughout the world.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

Distribution of Plasmopara viticola Causing Downy Mildew in Russian Far East Grapevines

Downy mildew is a severe disease that leads to significant losses in grape yields worldwide. It is caused by the oomycete Plasmopara viticola. The study of the distribution of this agent and the search for endophytic organisms that inhibit the growth of P. viticola are essential objectives to facilitate the transition to sustainable and high-yield agriculture, while respecting the environment. In this study, high-throughput sequencing of the ITS (ITS1f/ITS2 region) and 16S (V4 region) amplicons was employed to analyze 80 samples of leaves and stems from different grapevine species and cultivars grown in the Russian Far East (Vitis amurensis Rupr., Vitis coignetiae Pulliat, and several grapevine cultivars). The analysis revealed the presence of P. viticola in 53.75% of the grape samples. The pathogen P. viticola was not detected in V. amurensis samples collected near Vladivostok and Russky Island. Among the P. viticola-affected samples, only two (out of the eighty analyzed grape samples) from the Makarevich vineyard in Primorsky Krai exhibited disease symptoms, while the majority appeared visually healthy. We also found six distinct P. viticola ASVs in our metagenomic data. Based on phylogenetic analysis, we hypothesize that the P. viticola population in the Russian Far East may have originated from the invasive P. viticola clade aestivalis, which has spread around the world from North America. To identify putative microbial antagonists of P. viticola, a differential analysis of high-throughput sequencing data was conducted using the DESeq2 method to compare healthy and P. viticola-affected samples. The in silico analysis revealed an increased representation of certain taxa in healthy samples compared to P. viticola-affected ones: fungi—Kabatina sp., Aureobasidium sp., and Vishniacozyma sp.; bacteria—Hymenobacter spp., Sphingomonas spp., Massilia spp., Methylobacterium-Methylorubrum spp., and Chryseobacterium spp. This in-silico-obtained information on the potential microbial antagonists of P. viticola serves as a theoretical basis for the development of biocontrol agents for grapevine downy mildew.

Optimising high-throughput sequencing data analysis, from gene database selection to the analysis of compositional data: a case study on tropical soil nematodes

Optimising high-throughput sequencing data analysis, from gene database selection to the analysis of compositional data: a case study on tropical soil nematodes

Ds-Seq: An Integrated Pipeline for In Silico Small RNA Se-quence Analysis for Host-pathogen Interaction Studies

Plant-pathogen interactions activate molecular activities wherein the host defends the pathogen while the pathogen tries to suppress the plant response. Small RNAs (sRNAs) mediate major mechanisms, including post-transcriptional gene silencing, histone modification and DNA methylation by which plants respond to the presence of pathogens. Genome-wide profiling of host and pathogen sRNAs is therefore pivotal to uncovering the mechanisms underlying the host-pathogen interaction and mechanisms for host resistance. sRNA high throughput sequencing (HTS) data analysis often involves multiple stages/tools. Most necessary tools are accessible only through the command line, making it challenging for those without a high level of Unix/Linux skills. Furthermore, installation of some of these tools may become difficult due to dependencies and software version compatibility. We have developed an integrated open-source pipeline, Ds-Seq, for end-to-end in silico analysis of sRNA HTS data with improved reproducibility. The pipeline combines in-house scripts and public tools in a shell script, which can be invoked with a single command. The pipeline's usefulness has been demonstrated with testing on publicly available and published data from independent sRNA-seq datasets of host-pathogen interaction studies. Ds-Seq is available on GitHub, while a Docker image can be obtained from the Docker hub.Availability: Ds-Seq is freely available from the GitHub repository at https://github.com/CEPHAS-01/small-RNASeq.ngs and Docker hub with ID cephas/ds-seq (https://hub.docker.com/r/cephas/ds-seq).

Identification of BANF1 as a novel prognostic biomarker in gastric cancer and validation via in-vitro and in-vivo experiments.

Gastric cancer (GC) is a widespread malignancy characterized by a notably high incidence rate and an unfavorable prognosis. We conducted a meticulous analysis of GC high-throughput sequencing data downloaded from the Gene Expression Omnibus (GEO) repository to pinpoint distinctive genes associated with GC. Our investigation successfully identified three signature genes implicated in GC, with a specific focus on the barrier to autointegration factor 1 (BANF1), which exhibits elevated expression across various cancer types, including GC. Bioinformatic analysis has highlighted BANF1 as a prognostic indicator for patients with GC, with direct implications for immune cell infiltration. To gain a more comprehensive understanding of the significance of BANF1 in GC, we performed a series of in vitro experiments to confirm its high expression in GC tissues and cellular components. Intriguingly, the induction of BANF1 knockdown resulted in a marked attenuation of proliferation, migratory capacity, and invasive potential in GC cells. Moreover, our in vivo experiments using nude mouse models revealed a notable impediment in tumor growth following BANF1 knockdown. These insights underscore the feasibility of BANF1 as a novel therapeutic target for GC.

ExpoSeq: simplified analysis of high-throughput sequencing data from antibody discovery campaigns.

High-throughput sequencing (HTS) offers a modern, fast, and explorative solution to unveil the full potential of display techniques, like antibody phage display, in molecular biology. However, a significant challenge lies in the processing and analysis of such data. Furthermore, there is a notable absence of open-access user-friendly software tools that can be utilized by scientists lacking programming expertise. Here, we present ExpoSeq as an easy-to-use tool to explore, process, and visualize HTS data from antibody discovery campaigns like an expert while only requiring a beginner's knowledge. The pipeline is distributed via GitHub and PyPI, and it can either be installed as a package with pip or the user can choose to clone the repository.

Validation of High-Throughput Sequencing (HTS) for Routine Detection of Citrus Viruses and Viroids.

The management of plant diseases relies on the accurate identification of pathogens that requires a robust and validated tool in terms of specificity, sensitivity, repeatability, and reproducibility. High-throughput sequencing (HTS) has become the method of choice for virus detection when either a complete viral status of a plant is required in a single assay or if an unknown viral agent is expected. To ensure that the most accurate diagnosis is made from an HTS data analysis, a standardized protocol per pathosystem is required. This chapter presents a detailed protocol for the detection of viruses and viroids infecting citrus using HTS. The protocol describes all the steps from sample processing, nucleic acid extraction, and bioinformatic analyses validated to be an efficient method for detection in this pathosystem. The protocol also includes a section on citrus tristeza virus (CTV) genotype differentiation using HTS data.

Software pipelines for RNA-Seq, ChIP-Seq and germline variant calling analyses in common workflow language (CWL).

Background: Automating data analysis pipelines is a key requirement to ensure reproducibility of results, especially when dealing with large volumes of data. Here we assembled automated pipelines for the analysis of High-throughput Sequencing (HTS) data originating from RNA-Seq, ChIP-Seq and Germline variant calling experiments. We implemented these workflows in Common workflow language (CWL) and evaluated their performance by: i) reproducing the results of two previously published studies on Chronic Lymphocytic Leukemia (CLL), and ii) analyzing whole genome sequencing data from four Genome in a Bottle Consortium (GIAB) samples, comparing the detected variants against their respective golden standard truth sets. Findings: We demonstrated that CWL-implemented workflows clearly achieved high accuracy in reproducing previously published results, discovering significant biomarkers and detecting germline SNP and small INDEL variants. Conclusion: CWL pipelines are characterized by reproducibility and reusability; combined with containerization, they provide the ability to overcome issues of software incompatibility and laborious configuration requirements. In addition, they are flexible and can be used immediately or adapted to the specific needs of an experiment or study. The CWL-based workflows developed in this study, along with version information for all software tools, are publicly available on GitHub (https://github.com/BiodataAnalysisGroup/CWL_HTS_pipelines) under the MIT License. They are suitable for the analysis of short-read (such as Illumina-based) data and constitute an open resource that can facilitate automation, reproducibility and cross-platform compatibility for standard bioinformatic analyses.

Searching for sequencing signal anomalies associated with genome structural variations

Genomic structural variations (SVs) are one of the main sources of genetic diversity. Structural variants as mutagens may have a significant impact on human health and lead to hereditary diseases and cancers. Existing methods of finding structural variants are based on analysis of high-throughput sequencing data and despite significant progress in the development of the detection methods, there is still a need for improving the identification of structural variations with accuracy appropriate for use in a diagnostic procedure. Analysis of the signal of sequencing coverage (i.e., the number of sequencing fragments that aligned to every point of a genome) holds new potential for the design of approaches for structural variations discovery, and can be used as time-series analysis. Here, we present an approach for identification of patterns in the coverage signal. The method has been developed based on algorithms used for analysis of time series data, namely KNN (K-nearest neighbour) search algorithm and the SAX (Symbolic Aggregation Approximation) method. Using the rich dataset encompassing full genomes of 911 individuals with different ethnic backgrounds generated by the Human Genome Diversity Project initiative, we constructed generalized patterns of signal coverage in the vicinity of breakpoints corresponding to various structural variant types. Also, with the benefit of the SAX models of the motifs we developed a software package for fast detection of anomalies in the coverage signal.

The misregulation of mitochondria-associated genes caused by GAGA-factor lack promotes autophagic germ cell death in Drosophila testes.

The Drosophila GAGA-factor encoded by the Trithorax-like (Trl) gene is DNA-binding protein with unusually wide range of applications in diverse cell contexts. In Drosophila spermatogenesis, reduced GAGA expression caused by Trl mutations induces mass autophagy leading to germ cell death. In this work, we investigated the contribution of mitochondrial abnormalities to autophagic germ cell death in Trl gene mutants. Using a cytological approach, in combination with an analysis of high-throughput RNA sequencing (RNA-seq) data, we demonstrated that the GAGA deficiency led to considerable defects in mitochondrial ultrastructure, by causing misregulation of GAGA target genes encoding essential components of mitochondrial molecular machinery. Mitochondrial anomalies induced excessive production of reactive oxygen species and their release into the cytoplasm, thereby provoking oxidative stress. Changes in transcription levels of some GAGA-independent genes in the Trl mutants indicated that testis cells experience ATP deficiency and metabolic aberrations, that may trigger extensive autophagy progressing to cell death.

Discovery and Analyses of Caulimovirid-like Sequences in Upland Cotton (Gossypium hirsutum).

Analyses of Illumina-based high-throughput sequencing data generated during characterization of the cotton leafroll dwarf virus population in Mississippi (2020-2022) consistently yielded contigs varying in size (most frequently from 4 to 7 kb) with identical nucleotide content and sharing similarities with reverse transcriptases (RTases) encoded by extant plant pararetroviruses (family Caulimoviridiae). Initial data prompted an in-depth study involving molecular and bioinformatic approaches to characterize the nature and origins of these caulimovirid-like sequences. As a result, here, we report on endogenous viral elements (EVEs) related to extant members of the family Caulimoviridae, integrated into a genome of upland cotton (Gossypium hirsutum), for which we propose the provisional name "endogenous cotton pararetroviral elements" (eCPRVE). Our investigations pinpointed a ~15 kbp-long locus on the A04 chromosome consisting of head-to-head orientated tandem copies located on positive- and negative-sense DNA strands (eCPRVE+ and eCPRVE-). Sequences of the eCPRVE+ comprised nearly complete and slightly decayed genome information, including ORFs coding for the viral movement protein (MP), coat protein (CP), RTase, and transactivator/viroplasm protein (TA). Phylogenetic analyses of major viral proteins suggest that the eCPRVE+ may have been initially derived from a genome of a cognate virus belonging to a putative new genus within the family. Unexpectedly, an identical 15 kb-long locus composed of two eCPRVE copies was also detected in a newly recognized species G. ekmanianum, shedding some light on the relatively recent evolution within the cotton family.

Moderate body lipid accumulation in mice attenuated benzene-induced hematotoxicity via acceleration of benzene metabolism and clearance

Recent population and animal studies have revealed a correlation between fat content and the severity of benzene-induced hematologic toxicity. However, the precise impact of lipid deposition on benzene-induced hematotoxicity and the underlying mechanisms remain unclear. In this study, we established a mouse model with moderate lipid accumulation by subjecting the mice to an 8-week high-fat diet (45% kcal from fat, HFD), followed by 28-day inhalation of benzene at doses of 0, 1, 10, and 100 ppm. The results showed that benzene exposure caused a dose-dependent reduction of peripheral white blood cell (WBC) counts in both diet groups. Notably, this reduction was less pronounced in the HFD-fed mice, suggesting that moderate lipid accumulation mitigates benzene-related hematotoxicity. To investigate the molecular basis for this effect, we performed bioinformatics analysis of high-throughput transcriptome sequencing data, which revealed that moderate lipid deposition alters mouse metabolism and stress tolerance towards xenobiotics. Consistently, the expression of key metabolic enzymes, such as Cyp2e1 and Gsta1, were upregulated in the HFD-fed mice upon benzene exposure. Furthermore, we utilized a real-time exhaled breath detection technique to monitor exhaled benzene metabolites, and the results indicated that moderate lipid deposition enhanced metabolic activation and increased the elimination of benzene metabolites. Collectively, these findings demonstrate that moderate lipid deposition confers reduced susceptibility to benzene-induced hematotoxicity in mice, at least in part, by accelerating benzene metabolism and clearance.

Aerobiological Monitoring and Metabarcoding of Grass Pollen.

Grass pollen is one of the leading causes of pollinosis, affecting 10-30% of the world's population. The allergenicity of pollen from different Poaceae species is not the same and is estimated from moderate to high. Aerobiological monitoring is a standard method that allows one to track and predict the dynamics of allergen concentration in the air. Poaceae is a stenopalynous family, and thus grass pollen can usually be identified only at the family level with optical microscopy. Molecular methods, in particular the DNA barcoding technique, can be used to conduct a more accurate analysis of aerobiological samples containing the DNA of various plant species. This study aimed to test the possibility of using the ITS1 and ITS2 nuclear loci for determining the presence of grass pollen from air samples via metabarcoding and to compare the analysis results with the results of phenological observations. Based on the high-throughput sequencing data, we analyzed the changes in the composition of aerobiological samples taken in the Moscow and Ryazan regions for three years during the period of active flowering of grasses. Ten genera of the Poaceae family were detected in airborne pollen samples. The representation for most of them for ITS1 and ITS2 barcodes was similar. At the same time, in some samples, the presence of specific genera was characterized by only one sequence: either ITS1 or ITS2. Based on the analysis of the abundance of both barcode reads in the samples, the following order could describe the change with time in the dominant species in the air: Poa, Alopecurus, and Arrhenatherum in early mid-June, Lolium, Bromus, Dactylis, and Briza in mid-late June, Phleum, Elymus in late June to early July, and Calamagrostis in early mid-July. In most samples, the number of taxa found via metabarcoding analysis was higher compared to that in the phenological observations. The semi-quantitative analysis of high-throughput sequencing data well reflects the abundance of only major grass species at the flowering stage.

Sedimentary DNA and pigments show increasing abundance and toxicity of cyanoHABs during the Anthropocene

Abstract Cyanobacterial harmful algal blooms (cyanoHABs) are assumed to be increasing in abundance and toxicity, but comprehensive analysis of change through time is limited, in part, because some key taxa (e.g., Microcystis) leave ambiguous evidence of historical abundance and toxicity. Sedimentary DNA (sedDNA) can allow the reconstruction of the cyanobacteria community as well as the frequency of genes specific to cyanotoxin production, enabling us to determine which taxa are present and their potential for toxin‐production. Using a combination of droplet digital polymerase chain reaction (ddPCR) and high‐throughput sequencing (HTS), we quantified the abundance of cyanobacterial genes of known function and changes in cyanobacteria taxa from sedDNA over the last century in nine lakes along a gradient of lake size, depth and trophic state in Minnesota, U.S.A. Using ddPCR, we quantified genes associated with microcystin toxin‐producing potential (mcyE), total cyanobacteria (CYA, 16S rRNA) and the genus Microcystis (MICR, 16S rRNA). Using HTS on a subset of lakes, we investigated how the abundance of this toxin‐producing gene covaried with the cyanobacteria community composition. We also compared ddPCR and HTS data to fossil pigments, a well‐established palaeolimnological method used to track changes in primary producers over time. Our results showed a significant correlation between MICR and the quantity of mcyE gene and cyanobacterial taxa with known toxin‐production potential. The abundance of both genes likewise increased concomitantly through time. Community analyses of HTS data showed significant change in cyanobacterial communities commencing c. 1950 when major land‐use change in this region led to increased lake productivity, and c. 1990 when Dolichospermum and Microcystis genera increased in abundance, and the subtropical exotic cyanobacteria Raphidiopsis raciborskii and Sphaerospermopsis aphanizomenoides became abundant. Cyanobacteria pigment data reflected these changes only in deeper lakes, suggesting issues related to benthic production or biomarker preservation in shallower systems. This study provides evidence for historical development of increasingly toxic cyanoHABs across a diverse set of lakes and illustrates how sedDNA may help link changes in the cyanobacteria community to the expression of potentially toxic genes.

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.

High-throughput sequencing application in the detection and discovery of viruses associated with the regulated citrus leprosis disease complex.

Citrus leprosis (CiL) is one of the destructive emerging viral diseases of citrus in the Americas. Leprosis syndrome is associated with two taxonomically distinct groups of Brevipalpus-transmitted viruses (BTVs), that consist of positive-sense Cilevirus, Higrevirus, and negative-sense Dichorhavirus. The localized CiL symptoms observed in multiple citrus species and other alternate hosts indicates that these viruses might have originated from the mites and eventually adopted citrus as a secondary host. Genetic diversity in the genomes of viruses associated with the CiL disease complex have complicated current detection and diagnostic measures that prompted the application of High-Throughput Sequencing (HTS) protocols for improved detection and diagnosis. Two cileviruses are known to infect citrus, and among them only citrus leprosis virus C2 (CiLV-C2) hibiscus strain (CiLV-C2H) has been reported in hibiscus and passion fruit in the US. Based on our current CiL disease complex hypothesis, there is a high probability that CiL disease is associated with more viruses/strains that have not yet been identified but exist in nature. To protect the citrus industry, a Ribo-Zero HTS protocol was utilized for detection of cileviruses infecting three different hosts: Citrus spp., Swinglea glutinosa, and Hibiscus rosa-sinensis. Real-time RT-PCR assays were used to identify plants infected with CiLV-C2 or CiLV-C2H or both in mixed infection in all the above-mentioned plant genera. These results were further confirmed by bioinformatic analysis using HTS generated data. In this study, we utilized HTS assay in confirmatory diagnostics to screen BTVs infecting Dieffenbachia sp. (family: Araceae), Passiflora edulis (Passifloraceae), and Smilax auriculata (Smilacaceae). Through the implementation of HTS and downstream data analysis, we detected not only the known cileviruses in the studied hosts but also discovered a new strain of CiLV-C2 in hibiscus from Colombia. Phylogenetically, the new hibiscus strain is more closely related to CiLV-C2 than the known hibiscus strain, CiLV-C2H. We propose this strain to be named as CiLV-C2 hibiscus strain 2 (CiLV-C2H2). The findings from the study are critical for citrus growers, industry, regulators, and researchers. The possible movement of CiLV-C2H2 from hibiscus to citrus by the Brevipalpus spp. warrants further investigation.