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Small non-coding RNAs (sRNAs) regulate the synthesis of virulence factors and other pathogenic traits, which enables the bacteria to survive and proliferate after host infection. While high-throughput sequencing data have proved useful in identifying sRNAs from the intergenic regions (IGRs) of the genome, it remains a challenge to present a complete genome-wide map of the expression of the sRNAs. Moreover, existing methodologies necessitate multiple dependencies for executing their algorithm and also lack a targeted approach for the de novo sRNA identification. We developed an Isolation Forest algorithm-based method and the tool Prediction Of sRNAs using Isolation Forest for the de novo identification of sRNAs from available bacterial sRNA-seq data (http://posif.ibab.ac.in/). Using this framework, we predicted 1120 sRNAs and 46 small proteins in Mycobacterium tuberculosis. Besides, we highlight the context-dependent expression of novel sRNAs, their probable synthesis, and their potential relevance in stress response mechanisms manifested by M. tuberculosis.
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Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. In S. aureus, SprC is an antivirulent trans-acting sRNA known to base-pair with the major autolysin atl mRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we looked for its sRNA-RNA interactome and identified fourteen novel mRNA targets. In vitro biochemical investigations revealed that SprC binds two of them, czrB and deoD, and uses a single accessible region to regulate its targets, including Atl translation. Unlike Atl regulation, the characterization of the SprC-czrB interaction pinpointed a destabilization of czrAB co-transcript,leading to a decrease of the mRNA level that impaired CzrB Zinc efflux pump expression. On a physiological stand-point, we showed that SprC expression is detrimental to combat against Zinc toxicity. In addition, phagocyctosis assays revealed a significant, but moderate, increase of czrB mRNA level in a sprC-deleted mutant, indicating a functional link between SprC and czrB upon internalization in macrophages, and suggesting a role in resistance to both oxidative and Zinc burst. Altogether, our data uncover a novel pathway in which SprC is implicated, highlighting the multiple strategies employed by S. aureus to balance virulence using an RNA regulator.
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The purpose of this study was to evaluate the ability of Bacillus subtilis JATP3 to stimulate immune response and improve intestinal health in piglets during the critical weaning period. Twelve 28-day-old weaned piglets were randomly divided into two groups. One group was fed a basal diet, while the other group was fed a basal diet supplemented with B. subtilis JATP3 (1 × 109 CFU/mL; 10 mL) for 28 days. The results revealed a significant increase in the intestinal villus gland ratio of weaned piglets following the inclusion of B. subtilis JATP3 (P < 0.05). Inclusion of a probiotic supplement improve the intestinal flora of jejunum and ileum of weaned piglets. Metabolomics analysis demonstrated a notable rise in citalopram levels in the jejunum and ileum, along with elevated levels of isobutyric acid and isocitric acid in the ileum. The results of correlation analysis show that indicated a positive correlation between citalopram and microbial changes. Furthermore, the probiotic-treated group exhibited a significant upregulation in the relative expression of Claudin, Zonula Occludens 1 (ZO-1), and Interleukin 10 (IL-10) in the jejunum and ileum, while displaying a noteworthy reduction in the relative expression of Interleukin 1ß (IL-1ß). Overall, these findings suggest that B. subtilis JATP3 can safeguard intestinal health by modulating the structure of the intestinal microbiota and their metabolites, wherein citalopram might be a key component contributing to the therapeutic effects of B. subtilis JATP3.
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The molecular pathogenesis of Gram-negative bacteria remains a complex and incompletely understood phenomenon. Various factors are believed to contribute to the pathogenicity of these bacteria. One key mechanism utilized by Gram-negative bacteria is the production of outer membrane vesicles (OMVs), which are small spherical particles derived from the bacterial outer membrane. These OMVs are crucial in delivering virulence factors to the host, facilitating host-pathogen interactions. Within these OMVs, small regulatory RNAs (sRNAs) have been identified as important players in modulating the host immune response. One of the main challenges in studying OMVs and their cargo of sRNAs is the difficulty in isolating and purifying sufficient quantities of OMVs, as well as accurately predicting genuine sRNAs computationally. In this chapter, we present protocols aimed at overcoming these obstacles.
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Membrana Externa Bacteriana , Biologia Computacional , Pequeno RNA não Traduzido , Biologia Computacional/métodos , Pequeno RNA não Traduzido/genética , Membrana Externa Bacteriana/metabolismo , RNA Bacteriano/genética , Bactérias Gram-Negativas/genéticaRESUMO
Maize dwarf mosaic virus (MDMV) can significantly reduce the growth and development of susceptible varieties of sweet corn. The virus utilises the energy and reserve sources of plant cells to ensure its reproduction in the microspaces formed by cell membranes. Therefore, the severity of stress can be monitored by examining certain physiological changes, for example, changes in the degree of membrane damage caused by lipid peroxidation, as well as changes in the amount of photosynthetic pigments. The activation of antioxidant enzymes (e.g. ascorbate peroxidase, guaiacol peroxidase, glutathione reductase) and the accumulation of phenolic compounds with antioxidant properties can indirectly protect against the oxidative stress caused by the presence of the positive orientation, single-stranded RNA-virus. This study demonstrates the changes in these physiological processes in a sweet corn hybrid (Zea mays cv. saccharata var. Honey Koern.) susceptible to MDMV infection, and suggests that exogenous small RNA treatment can mitigate the damage caused by virus infection.
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Pseudomonas aeruginosa, an opportunistic bacterial pathogen of public health concern, is known for its metabolic versatility, adaptability in harsh environment, and pathogenic aggressiveness. P. aeruginosa relies on various regulatory networks modulated by small non-coding RNAs, which in turn influence different physiological traits such as metabolism, stress response, and pathogenesis. In this study, srbA sRNA has been shown to play a diverse role in regulating cellular metabolism and the production of different virulence factors in P. aeruginosa. srbA was found to control the TCA cycle, a key regulatory pathway for cellular metabolism and energy production, by regulating three main enzymes: citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-ketoglutarate dehydrogenase E1 subunit (sucA) at both the transcriptional and translational levels. By modulating the TCA cycle, srbA could help the bacteria to adapt nutritional stress by lowering energy consumption. Additionally, srbA has been found to differentially regulate production of various virulence factors such as rhamnolipid, elastase, LasA protease, and pyocyanin under both nutrient-rich and nutrient-limiting conditions. It could also influence motilities in P. aeruginosa, linked to biofilm formation and pathogenicity. Thus, srbA might hold a promise in the research area for identifying virulence pathways and developing novel therapeutic targets to combat the global pathogenic threat of P. aeruginosa.
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Staphylococcus aureus is a common colonizer of the skin and nares of healthy individuals, but also a major cause of severe human infections. During interaction with the host, pathogenic bacteria must adapt to a variety of adverse conditions including nutrient deprivation. In particular, they encounter severe iron limitation in the mammalian host through iron sequestration by haptoglobin and iron-binding proteins, a phenomenon called "nutritional immunity." In most bacteria, including S. aureus, the ferric uptake regulator (Fur) is the key regulator of iron homeostasis, which primarily acts as a transcriptional repressor of genes encoding iron acquisition systems. Moreover, Fur can control the expression of trans-acting small regulatory RNAs that play an important role in the cellular iron-sparing response involving major changes in cellular metabolism under iron-limiting conditions. In S. aureus, the sRNA IsrR is controlled by Fur, and most of its predicted targets are iron-containing proteins and other proteins related to iron metabolism and iron-dependent pathways. To characterize the IsrR targetome on a genome-wide scale, we combined proteomics-based identification of potential IsrR targets using S. aureus strains either lacking or constitutively expressing IsrR with an in silico target prediction approach, thereby suggesting 21 IsrR targets, of which 19 were negatively affected by IsrR based on the observed protein patterns. These included several Fe-S cluster- and heme-containing proteins, such as TCA cycle enzymes and catalase encoded by katA. IsrR affects multiple metabolic pathways connected to the TCA cycle as well as the oxidative stress response of S. aureus and links the iron limitation response to metabolic remodeling. In contrast to the majority of target mRNAs, the IsrR-katA mRNA interaction is predicted upstream of the ribosome binding site, and further experiments including mRNA half-life measurements demonstrated that IsrR, in addition to inhibiting translation initiation, can downregulate target protein levels by affecting mRNA stability.
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Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.
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In March of 2020, the World Health Organization declared COVID-19 a pandemic. The pandemic had unprecedented impacts on nurse anesthesia education delivery. The aim of this mixed methods study was to describe and quantify the personal and educational impacts of the COVID-19 pandemic on student registered nurse anesthetists (SRNAs). Three themes emerged from the qualitative arm of the study: 1) COVID-19 caused feelings of isolation, anxiety, and stress; 2) COVID-19 was a financial silver lining; and 3) COVID-19 changed nurse anesthesia education delivery and learning for SRNAs. The quantitative arm of the study revealed that SRNAs experienced anxiety, social isolation, and a sense of being overwhelmed. Almost half of the participants received federal stimulus money. Most participants reported an increase in distance education delivery and virtual simulation. This study summarizes the impact of the COVID-19 pandemic on SRNAs and how nurse anesthesia education was altered.
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COVID-19 , Enfermeiros Anestesistas , Estudantes de Enfermagem , COVID-19/epidemiologia , Humanos , Enfermeiros Anestesistas/educação , Estudantes de Enfermagem/psicologia , Feminino , Masculino , Adulto , Pandemias , SARS-CoV-2 , Educação a Distância , Ansiedade , Isolamento Social , Pessoa de Meia-IdadeRESUMO
Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.
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Doenças dos Peixes , Flagelos , Regulação Bacteriana da Expressão Gênica , Pseudomonas , Pequeno RNA não Traduzido , Pseudomonas/patogenicidade , Pseudomonas/genética , Pseudomonas/fisiologia , Virulência/genética , Animais , Doenças dos Peixes/microbiologia , Pequeno RNA não Traduzido/genética , Flagelos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Bacteriano/genética , Sistemas de Secreção Tipo III/genética , Bass , Infecções por Pseudomonas/imunologiaRESUMO
Bacterial chromosomal type I toxin-antitoxin systems consist of a small protein, typically under 60 amino acids, and a small RNA (sRNA) that represses toxin translation. These gene pairs have gained attention over the last decade for their contribution to antibiotic persistence and phage tolerance in bacteria. However, biological functions for many remain elusive as gene deletions often fail to produce an observable phenotype. For many pairs, it is still unknown when the toxin and/or antitoxin gene are natively expressed within the bacterium. We examined sequence conservation of three type I toxin-antitoxin systems, tisB/istR-1, shoB/ohsC, and zor/orz, in over 2,000 Escherichia coli strains, including pathogenic and commensal isolates. Using our custom database, we found that these gene pairs are widespread across E. coli and have expression potential via BLASTn. We identified an alternative, dominant sequence variant of TisB and confirmed that it is toxic upon overproduction. Additionally, analyses revealed a highly conserved sequence in the zorO mRNA untranslated region that is required for full toxicity. We further noted that over 30% of E. coli genomes contain an orz antitoxin gene only and confirmed its expression in a representative strain: the first confirmed report of a type I antitoxin without its cognate toxin. Our results add to our understanding of these systems, and our methodology is applicable for other type I loci to identify critical regulatory and functional features.IMPORTANCEChromosomal type I toxin-antitoxins are a class of genes that have gained increasing attention over the last decade for their roles in antibiotic persistence which may contribute to therapeutic failures. However, the control of many of these genes and when they function have remained elusive. We demonstrate that a simple genetic conservation-based approach utilizing free, publicly available data yields known and novel insights into the regulation and function of three chromosomal type I toxin-antitoxins in Escherichia coli. This study also provides a framework for how this approach could be applied to other genes of interest.
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Proteínas de Escherichia coli , Escherichia coli , Sistemas Toxina-Antitoxina , Escherichia coli/genética , Escherichia coli/metabolismo , Sistemas Toxina-Antitoxina/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
ArcZ is a small regulatory RNA conserved in Enterobacterales It is an Hfq-dependent RNA that is cleaved by RNase E in a processed form of 55-60 nucleotides. This processed form is highly conserved for controlling the expression of target mRNAs. ArcZ expression is induced by abundant oxygen levels and reaches its peak during the stationary growth phase. This control is mediated by the oxygen-responsive two-component system ArcAB, leading to the repression of arcZ transcription under low-oxygen conditions in most bacteria in which it has been studied. ArcZ displays multiple targets, and it can control up to 10% of a genome and interact directly with more than 300 mRNAs in Escherichia coli and Salmonella enterica ArcZ displays a multifaceted ability to regulate its targets through diverse mechanisms such as RNase recruitment, modulation of ribosome accessibility on the mRNA, and interaction with translational enhancing regions. By influencing stress response, motility, and virulence through the regulation of master regulators such as FlhDC or RpoS, ArcZ emerges as a major orchestrator of cell physiology within Enterobacterales.
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Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
INTRODUCTION: Salmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear. OBJECTIVES: This study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation. METHODS: Motility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter-reporter ß-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively. RESULTS: SaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter-reporter ß-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA. CONCLUSION: SaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.
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Next-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina's sequencing by synthesis method has long been essential for NGS but emerging technologies like Element Biosciences' sequencing by avidity (AVITI) represent a novel approach. It has been reported that AVITI offers improved signal-to-noise ratios and cost reductions. However, the method relies on rolling circle amplification which can be impacted by polymer size, raising questions about its efficacy sequencing small RNAs (sRNA) molecules including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and others that are crucial regulators of gene expression and involved in various biological processes. In addition, capturing capped small RNAs (csRNA-seq) has emerged as a powerful method to map active or "nascent" RNA polymerase II transcription initiation in tissues and clinical samples. Here, we report a new protocol for seamlessly sequencing short DNA fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus) and the bison (Bison bison) sRNA or csRNA sequencing libraries, augmenting the confidence in both approaches. Additionally, analysis of generated nascent transcription start sites (TSSs) data for cattle and bison revealed inaccuracies in their current genome annotations and highlighted the possibility and need to translate small RNA sequencing methodologies to livestock. Our accelerated and optimized protocol therefore bridges the advantages of AVITI sequencing and critical methods that rely on sequencing short DNA fragments.
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Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and confirmation with PCR and RT-PCR were used. Single and mixed infections with tomato mosaic virus (ToMV), tomato golden mosaic virus (ToGMoV), and pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR, as well as amino acid mutations in the ORFs encoding 126 KDa, 183 KDa, and MP of Mexican ToMV isolates, suggest that it is necessary to evaluate the capacity of these variants to overcome Tm-1, Tm-2, and Tm-22 resistance genes. This evaluation, along with the characterization of advanced lines using molecular markers linked to these resistant genes, will be addressed in future studies as part of the breeding strategy. This study emphasizes the importance of using HTS for accurate identification and characterization of plant viruses that naturally infect tomato germplasm based on the consensus genome sequences. This study provides crucial insights to select appropriate disease management strategies and resistance genes and guide breeding efforts toward the development of virus-resistant tomato varieties.
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Sequenciamento de Nucleotídeos em Larga Escala , Melhoramento Vegetal , Doenças das Plantas , Vírus de Plantas , Solanum lycopersicum , Doenças das Plantas/virologia , Solanum lycopersicum/virologia , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Vírus de Plantas/classificação , Genoma Viral/genética , Filogenia , Resistência à Doença/genética , RNA Viral/genéticaRESUMO
Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.
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Background: In recent years, research on exosomal miRNAs has provided new insights into exploring the mechanism of viral infection and disease prevention. This study aimed to investigate the serum exosomal miRNA expression profile of dengue-infected individuals through a community survey of dengue virus (DENV) infection. Methods: A seroprevalence study of 1253 healthy persons was first conducted to ascertain the DENV infection status in Baiyun District, Guangzhou. A total of 18 serum samples, including 6 healthy controls (HC), 6 asymptomatic DENV infections (AsymptDI), and 6 confirmed dengue fever patients (AcuteDI), were collected for exosome isolation and then sRNA sequencing. Through bioinformatics analysis, we discovered distinct serum exosomal miRNA profiles among the different groups and identified differentially expressed miRNAs (DEMs). These findings were further validated by qRT-PCR. Results: The community survey of DENV infection indicated that the DENV IgG antibody positivity rate among the population was 11.97 % in the study area, with asymptomatic infected individuals accounting for 93.06 % of the anti-DENV IgG positives. The age and Guangzhou household registration were associated with DENV IgG antibody positivity by logistic regression analysis. Distinct miRNA profiles were observed between healthy individuals and DENV infections. A total of 1854 miRNAs were identified in 18 serum exosome samples from the initial analysis of the sequencing data. Comparative analysis revealed 23 DEMs comprising 5 upregulated and 18 downregulated miRNAs in the DENV-infected group (mergedDI). In comparison to AcuteDI, 18 upregulated miRNAs were identified in AsymptDI. Moreover, functional enrichment of the predicted target genes of DEMs indicated that these miRNAs were involved in biological processes and pathways related to cell adhesion, focal adhesion, endocytosis, and ECM-receptor interaction. Eight DEMs were validated by qRT-PCR. Conclusion: The Baiyun District of Guangzhou exhibits a notable proportion of asymptomatic DENV infections as suggested in other research, highlighting the need for enhanced monitoring and screening of asymptomatic persons and the elderly. Differential miRNA expression among healthy, symptomatic and asymptomatic DENV-infected individuals suggests their potential as biomarkers for distinguishing DENV infection and offers new avenues of investigating the mechanisms underlying DENV asymptomatic infections.
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Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.
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Pequeno RNA não Traduzido , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Humanos , AnimaisRESUMO
Accurate annotation to single-nucleotide resolution of the transcribed regions in genomes is key to optimally analyse RNA-seq data, understand regulatory events and for the design of experiments. However, currently most genome annotations provided by GenBank generally lack information about untranslated regions. Additionally, information regarding genomic locations of non-coding RNAs, such as sRNAs, or anti-sense RNAs is frequently missing. To provide such information, diverse RNA-seq technologies, such as Rend-seq, have been developed and applied to many bacterial species. However, incorporating this vast amount of information into annotation files has been limited and is bioinformatically challenging, resulting in UTRs and other non-coding elements being overlooked or misrepresented. To overcome this problem, we present pyRAP (python Rend-seq Annotation Pipeline), a software package that analyses Rend-seq datasets to accurately resolve transcript boundaries genome-wide. We report the use of pyRAP to find novel transcripts, transcript isoforms, and RNase-dependent sRNA processing events. In Bacillus subtilis we uncovered 63 novel transcripts and provide genomic coordinates with single-nucleotide resolution for 2218 5'UTRs, 1864 3'UTRs and 161 non-coding RNAs. In Escherichia coli, we report 117 novel transcripts, 2429 5'UTRs, 1619 3'UTRs and 91 non-coding RNAs, and in Staphylococcus aureus, 16 novel transcripts, 664 5'UTRs, 696 3'UTRs, and 81 non-coding RNAs. Finally, we use pyRAP to produce updated annotation files for B. subtilis 168, E. coli K-12 MG1655, and S. aureus 8325 for use in the wider microbial genomics research community.