RESUMO
The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.
Assuntos
COVID-19 , Mutação , SARS-CoV-2 , Proteínas Virais Reguladoras e Acessórias , Virulência , COVID-19/virologia , Humanos , SARS-CoV-2/classificação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais Reguladoras e Acessórias/genética , Virulência/genética , Replicação Viral/genéticaRESUMO
Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in â¼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species (Brisavirus and Vientovirus) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCERedondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro, consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.
Assuntos
Infecções por Vírus de DNA/virologia , Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/metabolismo , Boca/virologia , Sistema Respiratório/virologia , Saliva/virologia , África/epidemiologia , Biodiversidade , Estado Terminal , Infecções por Vírus de DNA/epidemiologia , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Genoma Viral , Humanos , Metagenômica , Periodontite/virologia , Filogenia , Prevalência , População Rural , Estados Unidos/epidemiologia , Proteínas Virais/metabolismoRESUMO
Viruses in the family Redondoviridae have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Redondoviridae, which is available at ictv.global/report/redondoviridae.
Assuntos
Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/patogenicidade , Vírus de DNA/fisiologia , DNA Circular , DNA de Cadeia Simples , DNA Viral , Genoma Viral/genética , Humanos , Metagenômica , Fases de Leitura Aberta , Replicação ViralRESUMO
SUMMARY: High-throughput sequencing is a powerful technique for addressing biological questions. Grabseqs streamlines access to publicly available metagenomic data by providing a single, easy-to-use interface to download data and metadata from multiple repositories, including the Sequence Read Archive, the Metagenomics Rapid Annotation through Subsystems Technology server and iMicrobe. Users can download data and metadata in a standardized format from any number of samples or projects from a given repository with a single grabseqs command. AVAILABILITY AND IMPLEMENTATION: Grabseqs is an open-source tool implemented in Python and licensed under the MIT license. The source code is freely available at https://github.com/louiejtaylor/grabseqs, the Python Package Index and Anaconda Cloud repository. CONTACT: bushman@pennmedicine.upenn.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Metadados , Metagenoma , Metagenômica , SoftwareRESUMO
Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.
Assuntos
Antivirais/metabolismo , Vírus Defeituosos/genética , Genoma Viral , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/genética , Replicação Viral , Células A549 , Criança , Regulação Viral da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Transcrição Gênica , Interferência Viral , Proteínas ViraisRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1007707.].
RESUMO
BACKGROUND: Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. RESULTS: Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. CONCLUSIONS: Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.
Assuntos
Técnicas de Inativação de Genes/instrumentação , Técnicas de Inativação de Genes/métodos , Homologia de Genes/genética , Vírus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , Biologia Computacional/métodos , Bases de Dados Genéticas , Genes Virais/genética , Genoma Viral , SoftwareRESUMO
Redondoviruses are circular Rep-encoding single-stranded DNA (CRESS) viruses of high prevalence in healthy humans. Redondovirus abundance is increased in oro-respiratory samples from individuals with periodontitis, acute illness, and severe COVID-19. We investigated potential host cells supporting redondovirus replication in oro-respiratory samples and uncovered the oral amoeba Entamoeba gingivalis as a likely host. Redondoviruses are closely related to viruses of Entamoeba and contain reduced GC nucleotide content, consistent with Entamoeba hosts. Redondovirus and E. gingivalis co-occur in metagenomic data from oral disease and healthy human cohorts. When grown in xenic cultures with feeder bacteria, E. gingivalis was robustly positive for redondovirus RNA and DNA. A DNA proximity-ligation assay (Hi-C) on xenic culture cells showed enriched cross-linking of redondovirus and Entamoeba DNA, supporting E. gingivalis as the redondovirus host. While bacteria are established hosts for bacteriophages within the human virome, this work shows that eukaryotic commensals also contribute an abundant human-associated virus.
Assuntos
Bacteriófagos , COVID-19 , Entamoeba , Periodontite , Vírus , Humanos , Entamoeba/genética , BactériasRESUMO
Anelloviridae and Redondoviridae are virus families with small, circular, single-stranded DNA genomes that are common components of the human virome. Despite their small genome size of less than 5000 bases, they are remarkably successful - anelloviruses colonize over 90% of adult humans, while the recently discovered redondoviruses have been found at up to 80% prevalence in some populations. Anelloviruses are present in blood and many organs, while redondoviruses are found mainly in the ororespiratory tract. Despite their high prevalence, little is known about their biology or pathogenic potential. In this review, we discuss anelloviruses and redondoviruses and explore their enigmatic roles in human health and disease.
Assuntos
Anelloviridae , Adulto , Anelloviridae/genética , HumanosRESUMO
The ongoing COVID-19 pandemic has claimed more than 6 million lives and continues to test the world economy and healthcare systems. To combat this pandemic, the biological research community has shifted efforts to the development of medical countermeasures, including vaccines and therapeutics. However, to date, the only small molecules approved for the treatment of COVID-19 in the United States are the nucleoside analogue Remdesivir and the protease inhibitor Paxlovid, though multiple compounds have received Emergency Use Authorization and many more are currently being tested in human efficacy trials. One such compound, Apilimod, is being considered as a COVID-19 therapeutic in a Phase II efficacy trial. However, at the time of writing, there are no published efficacy data in human trials or animal COVID-19 models. Here we show that, while Apilimod and other PIKfyve inhibitors have potent antiviral activity in various cell lines against multiple human coronaviruses, these compounds worsen disease in a COVID-19 murine model when given prophylactically or therapeutically.
Assuntos
Tratamento Farmacológico da COVID-19 , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Modelos Animais de Doenças , Humanos , Camundongos , Pandemias , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de ProteasesRESUMO
We report the genome of a circular replication-associated protein (Rep)-encoding segmented or satellite virus, which we have provisionally named rengasvirus. In metagenomic studies of virus-enriched fractions, rengasvirus was detected widely, including in reagent-negative controls. We thus report this genome to help others recognize a probable contaminating sequence.
RESUMO
BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
Assuntos
COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sondas de Ácido Nucleico/genética , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e EspecificidadeRESUMO
Rationale: Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Objective: To define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes. Methods and Measurements: We examined 507 oropharyngeal, nasopharyngeal and endotracheal samples from 83 hospitalized COVID-19 patients, along with non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR, and immune features were characterized by lymphocyte/neutrophil (L/N) ratios and deep immune profiling of peripheral blood mononuclear cells (PBMC). Main Results: COVID-19 patients had upper respiratory microbiome dysbiosis, and greater change over time than critically ill patients without COVID-19. Diversity at the first time point correlated inversely with disease severity during hospitalization, and microbiome composition was associated with L/N ratios and PBMC profiles in blood. Intubated patients showed patient-specific and dynamic lung microbiome communities, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Conclusions: The respiratory tract microbiome and commensal virome are disturbed in COVID-19, correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, possible use as biomarkers, and role of bacterial and viral taxa identified here in COVID-19 pathogenesis.
RESUMO
Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.
Assuntos
Bactérias/classificação , Disbiose/microbiologia , Pulmão/microbiologia , Nasofaringe/microbiologia , Orofaringe/microbiologia , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anelloviridae/classificação , Anelloviridae/genética , Anelloviridae/isolamento & purificação , Bactérias/genética , Bactérias/isolamento & purificação , COVID-19/patologia , Feminino , Humanos , Contagem de Linfócitos , Masculino , Microbiota , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Índice de Gravidade de DoençaRESUMO
BACKGROUND: African populations provide a unique opportunity to interrogate host-microbe co-evolution and its impact on adaptive phenotypes due to their genomic, phenotypic, and cultural diversity. We integrate gut microbiome 16S rRNA amplicon and shotgun metagenomic sequence data with quantification of pathogen burden and measures of immune parameters for 575 ethnically diverse Africans from Cameroon. Subjects followed pastoralist, agropastoralist, and hunter-gatherer lifestyles and were compared to an urban US population from Philadelphia. RESULTS: We observe significant differences in gut microbiome composition across populations that correlate with subsistence strategy and country. After these, the variable most strongly associated with gut microbiome structure in Cameroonians is the presence of gut parasites. Hunter-gatherers have high frequencies of parasites relative to agropastoralists and pastoralists. Ascaris lumbricoides, Necator americanus, Trichuris trichiura, and Strongyloides stercoralis soil-transmitted helminths ("ANTS" parasites) significantly co-occur, and increased frequency of gut parasites correlates with increased gut microbial diversity. Gut microbiome composition predicts ANTS positivity with 80% accuracy. Colonization with ANTS, in turn, is associated with elevated levels of TH1, TH2, and proinflammatory cytokines, indicating an association with multiple immune mechanisms. The unprecedented size of this dataset allowed interrogation of additional questions-for example, we find that Fulani pastoralists, who consume high levels of milk, possess an enrichment of gut bacteria that catabolize galactose, an end product of lactose metabolism, and of bacteria that metabolize lipids. CONCLUSIONS: These data document associations of bacterial microbiota and eukaryotic parasites with each other and with host immune responses; each of these is further correlated with subsistence practices.
Assuntos
Fazendeiros/estatística & dados numéricos , Microbioma Gastrointestinal , Interações Hospedeiro-Parasita/imunologia , Nematoides/fisiologia , Carga Parasitária , Animais , Camarões , Dieta Paleolítica , Humanos , Estilo de Vida , Aprendizado de Máquina , Metagenoma , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Analysis of mixed microbial communities using metagenomic sequencing experiments requires multiple preprocessing and analytical steps to interpret the microbial and genetic composition of samples. Analytical steps include quality control, adapter trimming, host decontamination, metagenomic classification, read assembly, and alignment to reference genomes. RESULTS: We present a modular and user-extensible pipeline called Sunbeam that performs these steps in a consistent and reproducible fashion. It can be installed in a single step, does not require administrative access to the host computer system, and can work with most cluster computing frameworks. We also introduce Komplexity, a software tool to eliminate potentially problematic, low-complexity nucleotide sequences from metagenomic data. A unique component of the Sunbeam pipeline is an easy-to-use extension framework that enables users to add custom processing or analysis steps directly to the workflow. The pipeline and its extension framework are well documented, in routine use, and regularly updated. CONCLUSIONS: Sunbeam provides a foundation to build more in-depth analyses and to enable comparisons in metagenomic sequencing experiments by removing problematic, low-complexity reads and standardizing post-processing and analytical steps. Sunbeam is written in Python using the Snakemake workflow management software and is freely available at github.com/sunbeam-labs/sunbeam under the GPLv3.
Assuntos
Biologia Computacional/métodos , Metagenômica/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Análise de Dados , Mineração de Dados , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , SoftwareRESUMO
The global virome is largely uncharacterized but is now being unveiled by metagenomic DNA sequencing. Exploring the human respiratory virome, in particular, can provide insights into oro-respiratory diseases. Here, we use metagenomics to identify a family of small circular DNA viruses-named Redondoviridae-associated with human diseases. We first identified two redondovirus genomes from bronchoalveolar lavage samples from human lung donors. We then queried thousands of metagenomic samples and recovered 17 additional complete redondovirus genomes. Detections were exclusively in human samples and mostly from respiratory tract and oro-pharyngeal sites, where Redondoviridae was the second most prevalent eukaryotic DNA virus family. Redondovirus sequences were associated with periodontal disease, and abundances decreased with treatment. Some critically ill patients in a medical intensive care unit were found to harbor high levels of redondoviruses in respiratory samples. These results suggest that redondoviruses colonize human oro-respiratory sites and can bloom in several human disorders.
Assuntos
Estado Terminal , Infecções por Vírus de DNA/virologia , Vírus de DNA/classificação , Vírus de DNA/isolamento & purificação , Boca/virologia , Periodontite/virologia , Sistema Respiratório/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Vírus de DNA/genética , Vírus de DNA/patogenicidade , DNA Circular/genética , DNA Viral/genética , Feminino , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Historically, the human womb has been thought to be sterile in healthy pregnancies, but this idea has been challenged by recent studies using DNA sequence-based methods, which have suggested that the womb is colonized with bacteria. For example, analysis of DNA from placenta samples yielded small proportions of microbial sequences which were proposed to represent normal bacterial colonization. However, an analysis by our group showed no distinction between background negative controls and placenta samples. Also supporting the idea that the womb is sterile is the observation that germ-free mammals can be generated by sterile delivery of neonates into a sterile isolator, after which neonates remain germ-free, which would seem to provide strong data in support of sterility of the womb. RESULTS: To probe this further and to investigate possible placental colonization associated with spontaneous preterm birth, we carried out another study comparing microbiota in placenta samples from 20 term and 20 spontaneous preterm deliveries. Both 16S rRNA marker gene sequencing and shotgun metagenomic sequencing were used to characterize placenta and control samples. We first quantified absolute amounts of bacterial 16S rRNA gene sequences using 16S rRNA gene quantitative PCR (qPCR). As in our previous study, levels were found to be low in the placenta samples and indistinguishable from negative controls. Analysis by DNA sequencing did not yield a placenta microbiome distinct from negative controls, either using marker gene sequencing as in our previous work, or with shotgun metagenomic sequencing. Several types of artifacts, including erroneous read classifications and barcode misattribution, needed to be identified and removed from the data to clarify this point. CONCLUSIONS: Our findings do not support the existence of a consistent placental microbiome, in either placenta from term deliveries or spontaneous preterm births.
Assuntos
Bactérias/isolamento & purificação , Microbiota/genética , Placenta/microbiologia , Útero/microbiologia , Adulto , Bactérias/genética , DNA Bacteriano/genética , Feminino , Humanos , Gravidez , Nascimento Prematuro , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Nascimento a TermoRESUMO
Mammalian digit regeneration progresses through consistent stages: histolysis, inflammation, epidermal closure, blastema formation, and finally redifferentiation. What we do not yet know is how each stage can affect others. Questions of stage timing, tissue interactions, and microenvironmental states are becoming increasingly important as we look toward solutions for whole limb regeneration. This study focuses on the timing of epidermal closure which, in mammals, is delayed compared to more regenerative animals like the axolotl. We use a standard wound closure device, Dermabond (2-octyl cyanoacrylate), to induce earlier epidermal closure, and we evaluate the effect of fast epidermal closure on histolysis, blastema formation, and redifferentiation. We find that fast epidermal closure is reliant upon a hypoxic microenvironment. Additionally, early epidermal closure eliminates the histolysis stage and results in a regenerate that more closely replicates the amputated structure. We show that tools like Dermabond and oxygen are able to independently influence the various stages of regeneration enabling us to uncouple histolysis, wound closure, and other regenerative events. With this study, we start to understand how each stage of mammalian digit regeneration is controlled.