RESUMO
BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.
Assuntos
Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal , Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Viroma , Adulto , Idoso , Bacteriófagos , Clostridioides difficile , Enterocolite Pseudomembranosa/microbiologia , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/virologia , Humanos , Masculino , Metagenômica , Microviridae , Pessoa de Meia-Idade , Proteobactérias , Viroma/genéticaRESUMO
BACKGROUND & AIMS: Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. METHODS: C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. RESULTS: Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. CONCLUSIONS: Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.
Assuntos
Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteínas de Bactérias/imunologia , Toxina da Cólera/imunologia , Diarreia/diagnóstico , Diarreia/imunologia , Diarreia/microbiologia , Modelos Animais de Doenças , Disbiose/microbiologia , Feminino , Vida Livre de Germes , Humanos , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Pneumonia/diagnóstico , Pneumonia/imunologia , Pneumonia/microbiologia , Índice de Gravidade de DoençaRESUMO
Immunotherapies have led to the successful development of novel therapies for cancer. However, there is increasing concern regarding the adverse effects caused by non-tumor-specific immune responses. Here, we report an effective strategy to generate high-avidity tumor-antigen-specific CTLs, using Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) delivery. As a proof-of-principle demonstration, we selected the gp100 melanoma-associated tumor antigen, and cloned the gp100-specific high-avidity TCR from gp100-immunized mice. To enable rapid structural dissection of the TCR, we developed a 3D protein structure modeling system for the TCR/antigen-major histocompatibility complex (pMHC) interaction. Combining these technologies, we efficiently generated gp100-specific PD-1(-) CD8+ T cells, and demonstrated that the genetically engineered CD8+ T cells have high avidity against melanoma cells both in vitro and in vivo. Our methodology offers computational prediction of the TCR response, and enables efficient generation of tumor antigen-specific CD8+ T cells that can neutralize tumor-induced immune suppression leading to a potentially powerful cancer therapeutic.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Genes Reporter , Melanoma Experimental , Camundongos , Modelos Moleculares , Complexos Multiproteicos , Neoplasias/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/imunologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.
Assuntos
Fermentação , Lactobacillus delbrueckii/metabolismo , Probióticos/metabolismo , Streptococcus thermophilus/metabolismo , Iogurte/microbiologia , Animais , Intestinos/imunologia , Intestinos/microbiologia , Lactobacillus delbrueckii/genética , Lactobacillus delbrueckii/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Streptococcus thermophilus/genética , Streptococcus thermophilus/imunologiaRESUMO
Basic leucine zipper transcription factor Batf2 is poorly described, whereas Batf and Batf3 have been shown to play essential roles in dendritic cell, T cell, and B cell development and regulation. Batf2 was drastically induced in IFN-γ-activated classical macrophages (M1) compared with unstimulated or IL-4-activated alternative macrophages (M2). Batf2 knockdown experiments from IFN-γ-activated macrophages and subsequent expression profiling demonstrated important roles for regulation of immune responses, inducing inflammatory and host-protective genes Tnf, Ccl5, and Nos2. Mycobacterium tuberculosis (Beijing strain HN878)-infected macrophages further induced Batf2 and augmented host-protective Batf2-dependent genes, particularly in M1, whose mechanism was suggested to be mediated through both TLR2 and TLR4 by LPS and heat-killed HN878 (HKTB) stimulation experiments. Irf1 binding motif was enriched in the promoters of Batf2-regulated genes. Coimmunoprecipitation study demonstrated Batf2 association with Irf1. Furthermore, Irf1 knockdown showed downregulation of IFN-γ- or LPS/HKTB-activated host-protective genes Tnf, Ccl5, Il12b, and Nos2. Conclusively, Batf2 is an activation marker gene for M1 involved in gene regulation of IFN-γ-activated classical macrophages, as well as LPS/HKTB-induced macrophage stimulation, possibly by Batf2/Irf1 gene induction. Taken together, these results underline the role of Batf2/Irf1 in inducing inflammatory responses in M. tuberculosis infection.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Fator Regulador 1 de Interferon/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Mycobacterium/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/farmacologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Infecções por Mycobacterium/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ligação Proteica , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismoRESUMO
Motivation: Enhancers play critical roles in cell-type-specific transcriptional control. Despite the identification of thousands of candidate enhancers, unravelling their regulatory relationships with their target genes remains challenging. Therefore, computational approaches are needed to accurately infer enhancer-gene regulatory relationships. Results: In this study, we propose a new method, IVEA, that predicts enhancer-gene regulatory interactions by estimating promoter and enhancer activities. Its statistical model is based on the gene regulatory mechanism of transcriptional bursting, which is characterized by burst size and frequency controlled by promoters and enhancers, respectively. Using transcriptional readouts, chromatin accessibility, and chromatin contact data as inputs, promoter and enhancer activities were estimated using variational Bayesian inference, and the contribution of each enhancer-promoter pair to target gene transcription was calculated. Our analysis demonstrates that the proposed method can achieve high prediction accuracy and provide biologically relevant enhancer-gene regulatory interactions. Availability and implementation: The IVEA code is available on GitHub at https://github.com/yasumasak/ivea. The publicly available datasets used in this study are described in Supplementary Table S4.
RESUMO
The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as infectious disease, genetic disorder and drug dosage adjustment. Meanwhile, many of such technologies have complex reaction processes and often require a fine-tuned primer set where existing primer design tools are not sufficient. We have developed a primer selection system for one important primer, the turn-back primer (TP), which is commonly used in loop-mediated amplification (LAMP) and smart amplification process (SmartAmp). We chose 78 parameters related to the primer and target sequence, and explored their relationship to amplification speed using experimental data for 1344 primer combinations. We employed the least absolute shrinkage and selection operator (LASSO) method for parameter selection and estimation of their numerical coefficients. We subsequently evaluated our prediction model using additional independent experiments and compared to the LAMP primer design tool, Primer Explorer version4 (PE4). The evaluation showed that our approach yields a superior primer design in isothermal amplification and is robust against variations in the experimental setup. Our LASSO regression analysis revealed that availability of the 3'- and 5'-end of the primer are particularly important factors for efficient isothermal amplification. Our computer script is freely available at: http://gerg.gsc.riken.jp/TP_optimization/.
Assuntos
Primers do DNA/química , Técnicas de Amplificação de Ácido Nucleico , Humanos , Software , TemperaturaRESUMO
Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ~ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/. It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.
Assuntos
Benzotiazóis/química , DNA/química , Quinolinas/química , Timidina/química , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Desenho de Fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Termodinâmica , Temperatura de TransiçãoRESUMO
Transcriptional regulation by transcriptional regulatory factors (TRFs) of their target TRF genes is central to the control of gene expression. To study a static multi-tiered inter-TRF regulatory network in the human hepatoma cells, we have applied a Matrix RNAi approach in which siRNA knockdown and quantitative RT-PCR are used in combination on the same set of TRFs to determine their interdependencies. This approach focusing on several liver-enriched TRF families, each of which consists of structurally homologous members, revealed many significant regulatory relationships. These include the cross-talks between hepatocyte nuclear factors (HNFs) and the other TRF groups such as CCAAT/enhancer-binding proteins (CEBPs), retinoic acid receptors (RARs), retinoid receptors (RXRs) and RAR-related orphan receptors (RORs), which play key regulatory functions in human hepatocytes and liver. In addition, various multi-component regulatory motifs, which make up the complex inter-TRF regulatory network, were identified. A large part of the regulatory edges identified by the Matrix RNAi approach could be confirmed by chromatin immunoprecipitation. The resultant significant edges enabled us to depict the inter-TRF TRN forming an apparent regulatory hierarchy of (FOXA1, RXRA) --> TCF1 --> (HNF4A, ONECUT1) --> (RORC, CEBPA) as the main streamline.
Assuntos
Redes Reguladoras de Genes , Fígado/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
HIV-1 infected individuals under antiretroviral therapy can control viremia but often develop non-AIDS diseases such as cardiovascular and metabolic disorders. Gut microbiome dysbiosis has been indicated to be associated with progression of these diseases. Analyses of gut/fecal microbiome in individual regions are important for our understanding of pathogenesis in HIV-1 infections. However, data on gut/fecal microbiome has not yet been accumulated in West Africa. In the present study, we examined fecal microbiome compositions in HIV-1 infected adults in Ghana, where approximately two-thirds of infected adults are females. In a cross-sectional case-control study, age- and gender-matched HIV-1 infected adults (HIV+; n = 55) and seronegative controls (HIV-; n = 55) were enrolled. Alpha diversity of fecal microbiome in HIV+ was significantly reduced compared to HIV- and associated with CD4 counts. HIV+ showed reduction in varieties of bacteria including Faecalibacterium, the most abundant in seronegative controls, but enrichment of Proteobacteria. Ghanaian HIV+ exhibited enrichment of Dorea and Blautia; bacteria groups whose depletion has been reported in HIV-1 infected individuals in several other cohorts. Furthermore, HIV+ in our cohort exhibited a depletion of Prevotella, a genus whose enrichment has recently been shown in men having sex with men (MSM) regardless of HIV-1 status. The present study revealed the characteristics of dysbiotic fecal microbiome in HIV-1 infected adults in Ghana, a representative of West African populations.
Assuntos
Infecções por HIV , HIV-1 , Microbiota , Adulto , Estudos de Casos e Controles , Estudos Transversais , Disbiose , Feminino , Gana , Humanos , MasculinoRESUMO
BACKGROUND: There are an estimated 1·3-4·0 million cases of cholera and 20 000-140 000 cholera-related deaths worldwide each year. The rice-based cholera toxin B subunit (CTB) vaccine, MucoRice-CTB, is an oral candidate vaccine that does not require a cold chain, has shown efficacy in animal models, and could be of benefit in places where there is a paucity of medical infrastructure. We aim to assess the safety, tolerability, and immunogenicity of MucoRice-CTB in humans. METHODS: We did a double-blind, randomised, placebo-controlled, dose-escalation, phase 1 study at one centre in Tokyo, Japan. Eligible participants were healthy adult men with measurable serum and faecal antibodies against CTB at screening. Participants were excluded if they had allergy to rice; history of cholera or travellers' diarrhoea; poorly controlled constipation; abnormal results on hepatic, renal, or haematological screening tests; use of any over-the-counter drugs within 7 days before first administration; inability to use a medically acceptable means of contraception; or other reasons by medical judgment of the investigator. Three dose cohorts of participants were randomly assigned by block to receive oral MucoRice-CTB (1 g, 3 g, or 6 g) or placebo (1 g, 3 g, or 6 g), once every 2 weeks for 8 weeks (for a total of 4 doses). The dose groups were performed sequentially, and each dose cohort was completed before the higher dose cohort began. All medical staff, participants, and most trial staff were masked to treatment allocation. The primary outcomes were safety and tolerability, measured by 12-lead electrocardiogram; vital signs; haematology, biochemistry, and urinalysis; rice protein-specific serum IgE antibody concentration; and monitoring of adverse events. Participants were assessed at baseline and at 1, 2, 4, 6, 8, and 16 weeks after the first administration of vaccine or placebo. The safety analysis set included all participants enrolled in the trial who received at least one dose of the study drug or placebo and were compliant with good clinical practice. The full analysis population included all participants enrolled in the trial who received at least one dose of the study drug and for whom any data were obtained after the start of study drug administration. Meta-genomic analysis of study participants was performed using bacterial DNA from faecal samples before vaccination. This trial is registered with UMIN.ac.jp, UMIN000018001. FINDINGS: Between June 23, 2015, and May 31, 2016, 226 participants were recruited and assessed for eligibility. 166 participants were excluded based on health condition or schedule. We then randomly selected 60 male volunteers aged 20-40 years who were enrolled and assigned to MucoRice-CTB (10 participants assigned to 1 g, 10 participants assigned to 3 g, and 10 participants assigned to 6 g), or placebo (10 participants assigned to 1 g, 10 participants assigned to 3 g, and 10 participants assigned to 6 g). All participants received at least one dose of study drug or placebo and were included in the safety analyses. Two participants given MucoRice-CTB 3 g and one participant given MucoRice-CTB 6 g were lost to follow-up and excluded from the efficacy analysis. Serum CTB-specific IgG and IgA antibody concentrations in participants who received 6 g MucoRice-CTB increased significantly in both a time-dependent and dose-dependent manner compared with those in the placebo groups (p for interaction=0·002 for IgG, p=0·004 for IgA). Genome analysis of subjects' faeces before vaccination revealed that compared to non-responders, responders had a gut microbiota of higher diversity with the presence of Escherichia coli and Shigella spp. 28 (93%) of 30 participants who received MucoRice-CTB at any dose had at least one adverse event during the study period, compared with 30 (100%) of 30 participants given placebo. Grade 3 or higher adverse events were reported in four participants in the MucoRice-CTB group (5 events) and four participants in the placebo group (10 events). The most common serious adverse event was haemoglobin decreased (2 events in 2 participants in the pooled MucoRice-CTB group, 2 events in 2 participants in the placebo group; all grade 3). INTERPRETATION: Participants given MucoRice-CTB showed increased CTB-specific serum IgG and IgA antibody concentrations without inducing serious adverse events, indicating that MucoRice-CTB could be a safe and potent vaccine to prevent diarrhoeal disease. MucoRice-CTB induced neutralising antibodies against diarrhoeal toxins in a gut microbiota-dependent manner. A similar phase 1 trial will be done with participants of other ethnicities to substantiate our findings. FUNDING: Translational Research Acceleration Network Program of Japan Agency for Medical Research and Development; Ministry of Education, Culture, Sports, Science and Technology, Japan; Science and Technology Research Partnership for Sustainable Development; Grant-in-Aid for Scientific Research (S) (18H05280) (to H K) from the Japan Society for the Promotion of Science (JSPS); Grant-in-Aid for Young Scientists (B) (16K16144) (to Y K) from JSPS; Grant-in-Aid for Young Scientists (18K18148) (to Y K) from JSPS; Grant from International Joint Usage/Research Center (K3002), the Institute of Medical Science, University of Tokyo.
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COVID-19 , Cólera , Microbiota , Vacinas , Animais , Vacinas contra COVID-19 , Diarreia , Humanos , Imunogenicidade da Vacina , Imunoglobulina A , Imunoglobulina G , Masculino , SARS-CoV-2RESUMO
Recent studies have indicated an association between gut microbiome composition and various disorders, including infectious diseases. The composition of the microbiome differs among ethnicities and countries, possibly resulting in diversified interactions between host immunity and the gut microbiome. Characterization of baseline microbiome composition in healthy people is an essential step for better understanding of the biological interactions associated with individual populations. However, data on the gut/fecal microbiome have not been accumulated for individuals in West Africa. In the present study, we examined the fecal microbiome composition in healthy adults in Ghana. Toward this, 16S rRNA gene libraries were prepared using bacterial fractions derived from 55 Ghanaian adults, which were then subjected to next-generation sequencing. The fecal microbiome of the Ghanaian adults was dominated by Firmicutes (Faecalibacterium, Subdoligranulum, and Ruminococcaceae UCG-014), Proteobacteria (Escherichia-Shigella and Klebsiella), and Bacteroidetes (Prevotella 9 and Bacteroides), consistent with previous observations in African cohorts. Further, our analysis revealed differences in microbiome composition and a lower diversity of the fecal microbiome in the Ghanaian cohort compared with those reported in non-African countries. This is the first study to describe substantial fecal microbiome data obtained using high-throughput metagenomic tools on samples derived from a cohort in Ghana. The data may provide a valuable basis for determining the association between the fecal microbiome and progression of various diseases in West African populations.
Assuntos
Fezes/microbiologia , Microbioma Gastrointestinal/genética , Adulto , Bacteroidetes/genética , Estudos Transversais , Feminino , Firmicutes/genética , Gana , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metagenômica , Microbiota , Pessoa de Meia-Idade , Proteobactérias/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.
Assuntos
Corantes Fluorescentes/química , MicroRNAs/análise , Oligonucleotídeos/química , Fluorescência , MicroRNAs/química , Hibridização de Ácido NucleicoRESUMO
The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.
Assuntos
Bacteriófagos/genética , Clostridioides difficile/virologia , Endopeptidases/genética , Microbioma Gastrointestinal/genética , Terapia por Fagos/métodos , Prófagos/genética , Animais , Antibacterianos/farmacologia , Bacteriófagos/isolamento & purificação , Infecções por Clostridium/terapia , Modelos Animais de Doenças , Endopeptidases/farmacologia , Fezes/microbiologia , Feminino , Genoma Bacteriano , Genoma Viral , Humanos , Metagenoma , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de DNA , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
BACKGROUND: High-throughput real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) is a widely used technique in experiments where expression patterns of genes are to be profiled. Current stage technology allows the acquisition of profiles for a moderate number of genes (50 to a few thousand), and this number continues to grow. The use of appropriate normalization algorithms for qPCR-based data is therefore a highly important aspect of the data preprocessing pipeline. RESULTS: We present and evaluate two data-driven normalization methods that directly correct for technical variation and represent robust alternatives to standard housekeeping gene-based approaches. We evaluated the performance of these methods against a single gene housekeeping gene method and our results suggest that quantile normalization performs best. These methods are implemented in freely-available software as an R package qpcrNorm distributed through the Bioconductor project. CONCLUSION: The utility of the approaches that we describe can be demonstrated most clearly in situations where standard housekeeping genes are regulated by some experimental condition. For large qPCR-based data sets, our approaches represent robust, data-driven strategies for normalization.
Assuntos
Algoritmos , Biologia Computacional/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Bases de Dados Genéticas , Perfilação da Expressão GênicaRESUMO
Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either "strong" or "weak", depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with "strong tails". Unlike "strong tails", concentration change of FP with "weak tails" did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.
Assuntos
Primers do DNA/química , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , DNA/análise , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Conformação de Ácido Nucleico , Sensibilidade e Especificidade , Temperatura de TransiçãoRESUMO
T cell lymphopenia results in peripheral homeostatic expansion to maintain the T cell immune system, which is termed lymphopenia-induced proliferation (LIP). LIP is a potential risk for expanding autoreactive clones to become pathogenic in human and murine autoimmune diseases. However, the ontogeny of T cells that induce autoantibody production by autoreactive B cells in LIP remains unclear. Transfer of CD4+CD25- conventional T (Tc) cells into T-cell-deficient athymic nude mice has been previously reported as a LIP-induced autoimmune model which develops organ-specific autoimmune diseases and systemic antinuclear antibodies (ANAs). We show here that via LIP in this model, Tc cells proliferated and differentiated into PD-1+CXCR5-/dim B-helper T cells, which promoted splenic germinal center (GC) formation, provided help for autoantibody-producing B cells, and had distinctive features of follicular helper T (Tfh) cells except that they do not express high CXCR5. Intestinal microbiota were essential for their generation, since depletion of them in recipient mice by antibiotics resulted in a reduction of LIP-induced PD-1+CXCR5-/dim B-helper T cells and an amelioration of autoimmune responses. Our findings will contribute to the elucidation of the mechanism of lymphopenia-induced autoimmunity and autoantibody production, and will pave the way for microbiota-targeted novel therapeutic approaches to systemic autoimmune diseases.
Assuntos
Autoimunidade , Linfócitos B/imunologia , Microbioma Gastrointestinal , Linfopenia/imunologia , Linfopenia/microbiologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anticorpos Antinucleares , Formação de Anticorpos , Antígenos/metabolismo , Antígenos CD/metabolismo , Autoanticorpos/imunologia , Diferenciação Celular , Proliferação de Células , Fezes/microbiologia , Gastrite/tratamento farmacológico , Gastrite/imunologia , Gastrite/microbiologia , Centro Germinativo/metabolismo , Switching de Imunoglobulina , Linfopenia/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/patologiaRESUMO
Somatic mutation in human epidermal growth factor receptor-related 2 gene (HER2) is one of the driver mutations in lung cancer. HER2 mutations are found in about 2% of lung adenocarcinomas (ADCs). Previous reports have been based mainly on diagnostic screening by Sanger sequencing or next-generation sequencing (NGS); however, these methods are time-consuming and complicated. We developed a rapid, simple, sensitive mutation detection assay for detecting HER2 12 base pair-duplicated insertion mutation based on the Eprobe-mediated PCR method (Eprobe-PCR) and validated the sensitivity of this assay system for clinical diagnostics. We examined 635 tumor samples and analyzed HER2 mutations using the Eprobe-PCR method, NGS, and Sanger sequencing. In a serial dilution study, the Eprobe-PCR was able to detect mutant plasmid DNA when its concentration was reduced to 0.1% by mixing with wild-type DNA. We also confirmed amplification of the mutated plasmid DNA with only 10 copies per reaction. In ADCs, Eprobe-PCR detected the HER2 mutation in 2.02% (9/446), while Sanger sequencing detected it in 1.57% (7/446). Eprobe-PCR was able to detect the mutation in two samples that were undetectable by Sanger sequencing. All non-ADC samples were wild-type. There were no discrepancies between frozen and formalin-fixed paraffin-embedded tissues in the nine samples. HER2 mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.
Assuntos
Genes erbB-2/genética , Neoplasias Pulmonares/genética , Mutagênese Insercional/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.
Assuntos
Primers do DNA/genética , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pareamento Incorreto de Bases , Sondas de Oligonucleotídeos/genética , Taq Polimerase/metabolismoRESUMO
Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence 'Eprobe' capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05-0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.