Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) strains are the causative agents of severe foodborne diseases in both humans and animals. In this study, porcine pathogenic E. coli strains (n = 277) as well as porcine commensal strains (n = 188) were tested for their susceptibilities to 34 bacteriocin monoproducers to identify the most suitable bacteriocin types inhibiting porcine pathogens. Under in vitro conditions, the set of pathogenic E. coli strains was found to be significantly more susceptible to the majority of tested bacteriocins than commensal E. coli. Based on the production of bacteriocins with specific activity against pathogens, three potentially probiotic commensal E. coli strains of human origin were selected. These strains were found to be able to outcompete ETEC strains expressing F4 or F18 fimbriae in liquid culture and also decreased the severity and duration of diarrhea in piglets during experimental ETEC infection as well as pathogen numbers on the last day of in vivo experimentation. While the extents of the probiotic effect were different for each strain, the cocktail of all three strains showed the most pronounced beneficial effects, suggesting synergy between the tested E. coli strains. IMPORTANCE Increasing levels of antibiotic resistance among bacteria also increase the need for alternatives to conventional antibiotic treatment. Pathogenic Escherichia coli represents a major diarrheic infectious agent of piglets in their postweaning period; however, available measures to control these infections are limited. This study describes three novel E. coli strains producing antimicrobial compounds (bacteriocins) that actively inhibit a majority of toxigenic E. coli strains. The beneficial effect of three potentially probiotic E. coli strains was demonstrated under both in vitro and in vivo conditions. The novel probiotic candidates may be used as prophylaxis during piglets' postweaning period to overcome common infections caused by E. coli.
- MeSH
- bakteriální toxiny * metabolismus MeSH
- bakteriociny metabolismus terapeutické užití MeSH
- Escherichia coli * účinky léků genetika metabolismus MeSH
- faktory virulence genetika MeSH
- feces mikrobiologie MeSH
- infekce vyvolané Escherichia coli mikrobiologie prevence a kontrola veterinární MeSH
- nemoci prasat mikrobiologie prevence a kontrola MeSH
- prasata MeSH
- probiotika terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie veterinární MeSH
Common variable immunodeficiency (CVID) is a clinically and genetically heterogeneous disorder with inadequate antibody responses and low levels of immunoglobulins including IgA that is involved in the maintenance of the intestinal homeostasis. In this study, we analyzed the taxonomical and functional metagenome of the fecal microbiota and stool metabolome in a cohort of six CVID patients without gastroenterological symptomatology and their healthy housemates. The fecal microbiome of CVID patients contained higher numbers of bacterial species and altered abundance of thirty-four species. Hungatella hathewayi was frequent in CVID microbiome and absent in controls. Moreover, the CVID metagenome was enriched for low-abundance genes likely encoding nonessential functions, such as bacterial motility and metabolism of aromatic compounds. Metabolomics revealed dysregulation in several metabolic pathways, mostly associated with decreased levels of adenosine in CVID patients. Identified features have been consistently associated with CVID diagnosis across the patients with various immunological characteristics, length of treatment, and age. Taken together, this initial study revealed expansion of bacterial diversity in the host immunodeficient conditions and suggested several bacterial species and metabolites, which have potential to be diagnostic and/or prognostic CVID markers in the future.
- MeSH
- adenosin metabolismus MeSH
- běžná variabilní imunodeficience genetika mikrobiologie MeSH
- biodiverzita MeSH
- Clostridiaceae fyziologie MeSH
- dysbióza genetika mikrobiologie MeSH
- feces mikrobiologie MeSH
- homeostáza MeSH
- lidé MeSH
- metabolomika MeSH
- metagenom MeSH
- RNA ribozomální 16S genetika MeSH
- střevní mikroflóra genetika MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease.
- MeSH
- COVID-19 virologie MeSH
- CpG ostrůvky * MeSH
- genom virový MeSH
- lidé MeSH
- metylace DNA MeSH
- mutace * MeSH
- SARS-CoV-2 genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MOTIVATION: Transposable elements (TEs) in eukaryotes often get inserted into one another, forming sequences that become a complex mixture of full-length elements and their fragments. The reconstruction of full-length elements and the order in which they have been inserted is important for genome and transposon evolution studies. However, the accumulation of mutations and genome rearrangements over evolutionary time makes this process error-prone and decreases the efficiency of software aiming to recover all nested full-length TEs. RESULTS: We created software that uses a greedy recursive algorithm to mine increasingly fragmented copies of full-length LTR retrotransposons in assembled genomes and other sequence data. The software called TE-greedy-nester considers not only sequence similarity but also the structure of elements. This new tool was tested on a set of natural and synthetic sequences and its accuracy was compared to similar software. We found TE-greedy-nester to be superior in a number of parameters, namely computation time and full-length TE recovery in highly nested regions. AVAILABILITY AND IMPLEMENTATION: http://gitlab.fi.muni.cz/lexa/nested. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
MOTIVATION: G-quadruplex is a DNA or RNA form in which four guanine-rich regions are held together by base pairing between guanine nucleotides in coordination with potassium ions. G-quadruplexes are increasingly seen as a biologically important component of genomes. Their detection in vivo is problematic; however, sequencing and spectrometric techniques exist for their in vitro detection. We previously devised the pqsfinder algorithm for PQS identification, implemented it in C++ and published as an R/Bioconductor package. We looked for ways to optimize pqsfinder for faster and user-friendly sequence analysis. RESULTS: We identified two weak points where pqsfinder could be optimized. We modified the internals of the recursive algorithm to avoid matching and scoring many sub-optimal PQS conformations that are later discarded. To accommodate the needs of a broader range of users, we created a website for submission of sequence analysis jobs that does not require knowledge of R to use pqsfinder. AVAILABILITY AND IMPLEMENTATION: https://pqsfinder.fi.muni.cz, https://bioconductor.org/packages/pqsfinder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
- MeSH
- algoritmy MeSH
- G-kvadruplexy * MeSH
- genom MeSH
- RNA MeSH
- software MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Common Variable Immunodeficiency (CVID) is the most frequent symptomatic immune disorder characterized by reduced serum immunoglobulins. Patients often suffer from infectious and serious non-infectious complications which impact their life tremendously. The monogenic cause has been revealed in a minority of patients so far, indicating the role of multiple genes and environmental factors in CVID etiology. Using 16S and ITS rRNA amplicon sequencing, we analyzed the bacterial and fungal gut microbiota, respectively, in a group of 55 participants constituting of CVID patients and matched healthy controls including 16 case-control pairs living in the same household, to explore possible associations between gut microbiota composition and disease phenotype. We revealed less diverse and significantly altered bacterial but not fungal gut microbiota in CVID patients, which additionally appeared to be associated with a more severe disease phenotype. The factor of sharing the same household impacted both bacterial and fungal microbiome data significantly, although not as strongly as CVID diagnosis in bacterial assessment. Overall, our results suggest that gut bacterial microbiota is altered in CVID patients and may be one of the missing environmental drivers contributing to some of the symptoms and disease severity. Paired samples serving as controls will provide a better resolution between disease-related dysbiosis and other environmental confounders in future studies.
- MeSH
- Bacteria klasifikace genetika imunologie MeSH
- běžná variabilní imunodeficience imunologie mikrobiologie MeSH
- biodiverzita MeSH
- dospělí MeSH
- feces mikrobiologie MeSH
- houby klasifikace genetika imunologie MeSH
- imunoglobulin A krev imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mykobiom * MeSH
- senioři MeSH
- střevní mikroflóra * imunologie MeSH
- studie případů a kontrol MeSH
- zdraví rodiny MeSH
- zdravotní stav MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.
- MeSH
- alternativní sestřih * MeSH
- buněčné linie MeSH
- exony * MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- místa sestřihu RNA MeSH
- mutace * MeSH
- mutageneze MeSH
- protein přežití motorických neuronů 1 chemie genetika metabolismus MeSH
- RNA malá jaderná chemie genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Many studies have shown that guanine-rich DNA sequences form quadruplex structures (G4) in vitro but there is scarce evidence of guanine quadruplexes in vivo. The majority of potential quadruplex-forming sequences (PQS) are located in transposable elements (TEs), especially close to promoters within long terminal repeats of plant LTR retrotransposons. RESULTS: In order to test the potential effect of G4s on retrotransposon expression, we cloned the long terminal repeats of selected maize LTR retrotransposons upstream of the lacZ reporter gene and measured its transcription and translation in yeast. We found that G4s had an inhibitory effect on translation in vivo since "mutants" (where guanines were replaced by adenines in PQS) showed higher expression levels than wild-types. In parallel, we confirmed by circular dichroism measurements that the selected sequences can indeed adopt G4 conformation in vitro. Analysis of RNA-Seq of polyA RNA in maize seedlings grown in the presence of a G4-stabilizing ligand (NMM) showed both inhibitory as well as stimulatory effects on the transcription of LTR retrotransposons. CONCLUSIONS: Our results demonstrate that quadruplex DNA located within long terminal repeats of LTR retrotransposons can be formed in vivo and that it plays a regulatory role in the LTR retrotransposon life-cycle, thus also affecting genome dynamics.
- MeSH
- G-kvadruplexy * MeSH
- genetická transkripce MeSH
- genom rostlinný * MeSH
- koncové repetice * MeSH
- kukuřice setá genetika růst a vývoj metabolismus MeSH
- reportérové geny * MeSH
- retroelementy * MeSH
- Saccharomyces cerevisiae genetika růst a vývoj MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Motivation: G-quadruplexes (G4s) are one of the non-B DNA structures easily observed in vitro and assumed to form in vivo. The latest experiments with G4-specific antibodies and G4-unwinding helicase mutants confirm this conjecture. These four-stranded structures have also been shown to influence a range of molecular processes in cells. As G4s are intensively studied, it is often desirable to screen DNA sequences and pinpoint the precise locations where they might form. Results: We describe and have tested a newly developed Bioconductor package for identifying potential quadruplex-forming sequences (PQS). The package is easy-to-use, flexible and customizable. It allows for sequence searches that accommodate possible divergences from the optimal G4 base composition. A novel aspect of our research was the creation and training (parametrization) of an advanced scoring model which resulted in increased precision compared to similar tools. We demonstrate that the algorithm behind the searches has a 96% accuracy on 392 currently known and experimentally observed G4 structures. We also carried out searches against the recent G4-seq data to verify how well we can identify the structures detected by that technology. The correlation with pqsfinder predictions was 0.622, higher than the correlation 0.491 obtained with the second best G4Hunter. Availability and implementation: http://bioconductor.org/packages/pqsfinder/ This paper is based on pqsfinder-1.4.1. Contact: lexa@fi.muni.cz. Supplementary information: Supplementary data are available at Bioinformatics online.
- MeSH
- algoritmy MeSH
- G-kvadruplexy * MeSH
- genomika metody MeSH
- sekvenční analýza DNA metody MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
