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1.
J Immunother Cancer ; 7(1): 193, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337439

RESUMO

BACKGROUND: Checkpoint-blockade immunotherapy targeting programmed cell death protein 1 (PD-1) has recently shown promising efficacy in hepatocellular carcinoma (HCC). However, the factors affecting and predicting the response to anti-PD-1 immunotherapy in HCC are still unclear. Herein, we report the dynamic variation characteristics and specificities of the gut microbiome during anti-PD-1 immunotherapy in HCC using metagenomic sequencing. RESULTS: Fecal samples from patients responding to immunotherapy showed higher taxa richness and more gene counts than those of non-responders. For dynamic analysis during anti-PD-1 immunotherapy, the dissimilarity of beta diversity became prominent across patients as early as Week 6. In non-responders, Proteobacteria increased from Week 3, and became predominant at Week 12. Twenty responder-enriched species, including Akkermansia muciniphila and Ruminococcaceae spp., were further identified. The related functional genes and metabolic pathway analysis, such as carbohydrate metabolism and methanogenesis, verified the potential bioactivities of responder-enriched species. CONCLUSIONS: Gut microbiome may have a critical impact on the responses of HCC patients treated with anti-PD-1 immunotherapy. The dynamic variation characteristics of the gut microbiome may provide early predictions of the outcomes of immunotherapy in HCC, which is critical for disease-monitoring and treatment decision-making.

2.
Sci Rep ; 9(1): 3453, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837612

RESUMO

Intestinal microbiota plays a crucial role in immune development and disease progression in mammals from birth onwards. The gastrointestinal tract of newborn mammals is rapidly colonized by microbes with tremendous biomass and diversity. Understanding how this complex of segmental communities evolves in different gastrointestinal sites over time has great biological significance and medical implications. However, most previous reports examining intestinal microbiota have focused on fecal samples, a strategy that overlooks the spatial microbial dynamics in different intestinal segments. Using intestinal digesta from six intestinal segments (duodenum, jejunum, ileum, cecum, colon and rectum) of newborn piglets, we herein conducted a large-scale 16S rRNA gene sequencing-based study to characterize the segmental dynamics of porcine gut microbiota at eight postnatal intervals (days 1, 7, 14, 21, 28, 35, 120 and 180). A total of 4,465 OTUs were obtained and showed that the six intestinal segments could be divided into three parts; in the duodenum-jejunum section, the most abundant genera included Lactobacillus and Bacteroides; in the ileum, Fusobacterium and Escherichia; and in the cecum-rectum section, Prevotella. Although the microbial communities of the piglets were similar among the six intestinal segments on postnatal day 1, they evolved and quickly differentiated at later intervals. An examination of time-dependent alterations in the dominant microbes revealed that the microbiome in the large intestine was very different from and much more stable than that in the small intestine. The gut microbiota in newborn piglets exhibited apparent temporal and spatial variations in different intestinal segments. The database of gut microbes in piglets could be a referable resource for future studies on mammalian gut microbiome development in early host growth phases.

4.
Genome Biol ; 18(1): 142, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28750650

RESUMO

BACKGROUND: The assessment and characterization of the gut microbiome has become a focus of research in the area of human autoimmune diseases. Ankylosing spondylitis is an inflammatory autoimmune disease and evidence showed that ankylosing spondylitis may be a microbiome-driven disease. RESULTS: To investigate the relationship between the gut microbiome and ankylosing spondylitis, a quantitative metagenomics study based on deep shotgun sequencing was performed, using gut microbial DNA from 211 Chinese individuals. A total of 23,709 genes and 12 metagenomic species were shown to be differentially abundant between ankylosing spondylitis patients and healthy controls. Patients were characterized by a form of gut microbial dysbiosis that is more prominent than previously reported cases with inflammatory bowel disease. Specifically, the ankylosing spondylitis patients demonstrated increases in the abundance of Prevotella melaninogenica, Prevotella copri, and Prevotella sp. C561 and decreases in Bacteroides spp. It is noteworthy that the Bifidobacterium genus, which is commonly used in probiotics, accumulated in the ankylosing spondylitis patients. Diagnostic algorithms were established using a subset of these gut microbial biomarkers. CONCLUSIONS: Alterations of the gut microbiome are associated with development of ankylosing spondylitis. Our data suggest biomarkers identified in this study might participate in the pathogenesis or development process of ankylosing spondylitis, providing new leads for the development of new diagnostic tools and potential treatments.


Assuntos
Doenças Autoimunes/microbiologia , Disbiose/microbiologia , Microbioma Gastrointestinal/genética , Genes Bacterianos , Metagenômica/métodos , Espondilite Anquilosante/microbiologia , Adolescente , Adulto , Idoso , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Bacteroides/classificação , Bacteroides/genética , Bacteroides/isolamento & purificação , Bifidobacterium/classificação , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , Biomarcadores/metabolismo , Estudos de Casos e Controles , Disbiose/imunologia , Disbiose/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Prevotella/classificação , Prevotella/genética , Prevotella/isolamento & purificação , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/patologia
6.
Sci Rep ; 6: 34826, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27708392

RESUMO

The human gut microbiota varies considerably among world populations due to a variety of factors including genetic background, diet, cultural habits and socioeconomic status. Here we characterized 110 healthy Mongolian adults gut microbiota by shotgun metagenomic sequencing and compared the intestinal microbiome among Mongolians, the Hans and European cohorts. The results showed that the taxonomic profile of intestinal microbiome among cohorts revealed the Actinobaceria and Bifidobacterium were the key microbes contributing to the differences among Mongolians, the Hans and Europeans at the phylum level and genus level, respectively. Metagenomic species analysis indicated that Faecalibacterium prausnitzii and Coprococcus comeswere enrich in Mongolian people which might contribute to gut health through anti-inflammatory properties and butyrate production, respectively. On the other hand, the enriched genus Collinsella, biomarker in symptomatic atherosclerosis patients, might be associated with the high morbidity of cardiovascular and cerebrovascular diseases in Mongolian adults. At the functional level, a unique microbial metabolic pathway profile was present in Mongolian's gut which mainly distributed in amino acid metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism, glycan biosynthesis and metabolism. We can attribute the specific signatures of Mongolian gut microbiome to their unique genotype, dietary habits and living environment.


Assuntos
Microbioma Gastrointestinal/fisiologia , Actinobacteria/genética , Actinobacteria/fisiologia , Adulto , Grupo com Ancestrais do Continente Asiático , Bifidobacterium/genética , Bifidobacterium/fisiologia , Butiratos/metabolismo , Doenças Cardiovasculares/microbiologia , Comportamento Alimentar , Microbioma Gastrointestinal/genética , Humanos , Metagenoma , Mongólia
7.
Acta Biochim Biophys Sin (Shanghai) ; 48(11): 1050-1057, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27649890

RESUMO

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.


Assuntos
Bombyx/parasitologia , Nosema/patogenicidade , Ricina/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Ricina/química , Ricina/genética
8.
Acta Biochim Biophys Sin (Shanghai) ; 48(3): 246-56, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26837419

RESUMO

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori. Germination, an indispensible process through which microsporidia infect the host cells, is regarded as a key developmental turning point for microsporidia from dormant state to reproduction state. Thus, elucidating the transcriptome changes before and after germination is crucial for parasite control. However, the molecular basis of germination of microsporidia remains unknown. To investigate this germination process, the transcriptome of N. bombycis ungerminated spores and germinated spores were sequenced and analyzed. More than 60 million high-quality transcript reads were generated from these two groups using RNA-Seq technology. After assembly, 2756 and 2690 unigenes were identified, respectively, and subsequently annotated based on known proteins. After analysis of differentially expressed genes, 66 genes were identified to be differentially expressed (P ≤ 0.05) between these two groups. A protein phosphatase-associated gene was first identified to be significantly up-regulated as determined by RNA-Seq and immunoblot analysis, indicating that dephosphorylation might potentially contribute to microsporidia germination. The DEGs that encode proteins involved in glycometabolism, spore wall proteins and ricin B lectin of N. bombycis were also analyzed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed genes responsible for some specific biological functions and processes. The datasets generated in this study provide a basic characterization of the transcriptome changes in N. bombycis during germination. The analysis of transcriptome data and identification of certain functional genes which are robust candidate genes related to germination will help to provide a deep understanding of spore germination and invasion.


Assuntos
Germinação , Nosema/fisiologia , Esporos Fúngicos , Transcriptoma , Genes Fúngicos , Nosema/genética
9.
J Microbiol Methods ; 120: 72-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26658327

RESUMO

Pebrine disease is the only mandatory quarantine item in sericultural production due to its destructive consequences. So far, the mother moth microscopic examination method established by Pasteur (1870) remains the only detection method for screening for the causative agent Nosema bombycis (N. bombycis). Because pebrine is a horizontal and vertical transmission disease, it is better to inspect silkworm eggs and newly hatched larvae to investigate the infection rate, vertical transmission rate and spore load of the progenies. There is a rising demand for a more direct, effective and accurate detection approach in the sericultural industry. Here, we developed a molecular detection approach based on real-time quantitative PCR (qPCR) for pebrine inspection in single silkworm eggs and newly hatched larvae. Targeting the small-subunit rRNA gene of N. bombycis, this assay showed high sensitivity and reproducibility. Ten spores in a whole sample or 0.1 spore DNA (1 spore DNA represents the DNA content of one N. bombycis spore) in a reaction system was estimated as the detection limit of the isolation and real-time qPCR procedure. Silkworm egg tissues impact the detection sensitivity but are not significant in single silkworm egg detection. Of 400 samples produced by infected moths, 167 and 195 were scored positive by light microscopy and real-time qPCR analysis, respectively. With higher accuracy and the potential capability of high-throughput screening, this method is anticipated to be adaptable for pebrine inspection and surveillance in the sericultural industry. In addition, this method can be applied to ecology studies of N. bombycis-silkworm interactions due to its quantitative function.


Assuntos
Bombyx/microbiologia , Nosema/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Primers do DNA , DNA Fúngico/análise , DNA Fúngico/isolamento & purificação , Feminino , Genes de RNAr , Microbiologia Industrial/métodos , Larva/microbiologia , Masculino , Nosema/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Esporos Fúngicos/genética , Esporos Fúngicos/isolamento & purificação
10.
Acta Biochim Biophys Sin (Shanghai) ; 47(9): 696-702, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26188202

RESUMO

Nosema bombycis (N. bombycis, Nb) is a fungus-related and obligate intracellular parasite that causes chronic pebrine disease in the silkworm. After infecting the host, spores obtain energy from host cells and survive for several days. This symbiosis between the pathogen and the host cell suggests that N. bombycis prevents apoptosis and reactive oxygen species (ROS) production of host cells to create the optimal environmental conditions for its growth and development. In this study, different methods were used to prove that N. bombycis suppressed apoptosis in BmN cells. Flow cytometry analysis results showed that spores suppressed apoptosis of BmN cells at 2 and 5 days after infection (P < 0.05). Compared with actinomycin D (ActD) treatment, apoptosis of BmN cells was apparently reduced after spore infection (P < 0.01). Forty-eight hours after infection, the ROS production of BmN cells was down-regulated compared with that after ActD treatment for 6 h. Furthermore, N. bombycis prevented the formation of apoptosomes by down-regulating the expression of apaf-1 and cytochrome C. In addition, N. bombycis also up-regulated the expression of buffy. Western blot analysis demonstrated that spores decreased the level of host cytochrome C at 48 and 98 h post infection. Thus, our results suggested that N. bombycis inhibited the mitochondrial apoptotic pathway of the host cells to create an optimal environment for its own survival.


Assuntos
Apoptose/fisiologia , Nosema/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Bombyx , Células Cultivadas , Citocromos c/metabolismo , Dactinomicina/farmacologia , Citometria de Fluxo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo
11.
Acta Biochim Biophys Sin (Shanghai) ; 46(11): 982-90, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25267721

RESUMO

Nosema bombycis (N. bombycis, Nb) is an obligate intracellular parasite, which can cause pebrine disease in the silkworm. To investigate the effects of N. bombycis infection on the host cells, proteomes from BmN cells that had or had not been infected with N. bombycis at different infection stages were characterized with two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, which identified 24 differentially expressed host proteins with significant intensity differences (P < 0.05) at least at one time point in mock- and N. bombycis infected cells. Notably, gene ontology analyses showed that these proteins are involved in many important biological reactions. During the infection phase, proteins involved in energy metabolism and oxidative stress had up-regulated expression. Two proteins participated in ubiquitin-dependent protein catabolic process had down-regulated expression. Quantitative real-time polymerase chain reaction was used to analyze the transcriptional profiles of these identified proteins. Taken together, the abundance changes, putative functions, and participation in biological reactions for the identified proteins produce a host-responsive protein model in N. bombycis-infected BmN cells. These findings further our knowledge about the effect of energy defect parasites on the host cells.


Assuntos
Bombyx/metabolismo , Bombyx/microbiologia , Proteínas de Insetos/metabolismo , Microsporidiose/metabolismo , Nosema/patogenicidade , Animais , Bombyx/genética , Eletroforese em Gel Bidimensional , Metabolismo Energético , Perfilação da Expressão Gênica , Genes de Insetos , Interações Hospedeiro-Patógeno/genética , Proteínas de Insetos/genética , Microscopia Eletrônica de Transmissão , Microsporidiose/genética , Nosema/ultraestrutura , Estresse Oxidativo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Appl Microbiol Biotechnol ; 95(3): 697-705, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22476262

RESUMO

The 5' untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.


Assuntos
Biossíntese de Proteínas , Vírus de RNA/genética , Animais , Baculoviridae/genética , Bombyx/virologia , Linhagem Celular , Vetores Genéticos , Ribossomos/metabolismo
13.
Parasitology ; 138(9): 1102-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21756420

RESUMO

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.


Assuntos
Bombyx/parasitologia , Proteínas Fúngicas , Nosema/fisiologia , Ovário/parasitologia , Fagócitos/parasitologia , Esporos Fúngicos/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Antifúngicos/análise , Bombyx/citologia , Bombyx/metabolismo , Parede Celular/química , Células Cultivadas , Centrifugação com Gradiente de Concentração , Eletroforese em Gel Bidimensional , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Parasita , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Nosema/química , Ovário/citologia , Ovário/metabolismo , Fagócitos/citologia , Fagócitos/metabolismo , Fagocitose/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esporos Fúngicos/química
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