RESUMO
BACKGROUND AND AIMS: Bile acids are hepatic metabolites and have many properties considered to be relevant to the pathophysiology of NAFLD. Circulating levels of the intestinal microbiome-modified bile acid deoxycholate are increased in cirrhosis. APPROACH AND RESULTS: To further elucidate the role of bile acids and intestinal microbiota linked to bile acids in progressively severe NAFLD, a multiomic study of feces including 16S rRNA sequencing, microbial transcriptomics and metabolomics was performed in a cohort with varying phenotypes of NAFLD. Several bile acids of microbial origin derived from deoxycholic acid (DCA) (glycodeoxycholate, 7-ketodeoxycholic acid, dehydrocholic acid) increased with disease activity and fibrosis stage. These were linked to increased expression of microbial bile salt hydrolase, bile acid operon (BaiCD) and hydroxysteroid dehydrogenases (hdhA) required for DCA and downstream metabolite synthesis providing a mechanistic basis for altered bile acid profiles with disease progression. Bacteroidetes and several genera of Lachnospiraceae family containing DCA generating genes increased with increasing disease severity, whereas several potentially beneficial microbes sensitive to antibacterial effects of DCA e.g., Ruminococcaceae were decreased. The clinical relevance of these data was confirmed in an independent cohort enrolled in a clinical trial for NASH where at entry DCA and its conjugates were associated with advanced fibrosis. In patients treated with placebo, DCA declined in those with fibrosis regression and increased in those with fibrosis progression. DCA rose further in those with compensated cirrhosis when they experienced decompensation. CONCLUSIONS: These findings demonstrate a role for bile acids and the bile acid dependent microbiome in the development and progression of NAFLD and set the stage to leverage these findings for NASH biomarker development and for therapeutics.
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Humanos , Ácidos e Sais Biliares/farmacologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , RNA Ribossômico 16S , Cirrose HepáticaRESUMO
BACKGROUND: Shotgun metagenomic sequencing reveals the potential in microbial communities. However, lower-cost 16S ribosomal RNA (rRNA) gene sequencing provides taxonomic, not functional, observations. To remedy this, we previously introduced Piphillin, a software package that predicts functional metagenomic content based on the frequency of detected 16S rRNA gene sequences corresponding to genomes in regularly updated, functionally annotated genome databases. Piphillin (and similar tools) have previously been evaluated on 16S rRNA data processed by the clustering of sequences into operational taxonomic units (OTUs). New techniques such as amplicon sequence variant error correction are in increased use, but it is unknown if these techniques perform better in metagenomic content prediction pipelines, or if they should be treated the same as OTU data in respect to optimal pipeline parameters. RESULTS: To evaluate the effect of 16S rRNA sequence analysis method (clustering sequences into OTUs vs amplicon sequence variant error correction into amplicon sequence variants (ASVs)) on the ability of Piphillin to predict functional metagenomic content, we evaluated Piphillin-predicted functional content from 16S rRNA sequence data processed through OTU clustering and error correction into ASVs compared to corresponding shotgun metagenomic data. We show a strong correlation between metagenomic data and Piphillin-predicted functional content resulting from both 16S rRNA sequence analysis methods. Differential abundance testing with Piphillin-predicted functional content exhibited a low false positive rate (< 0.05) while capturing a large fraction of the differentially abundant features resulting from corresponding metagenomic data. However, Piphillin prediction performance was optimal at different cutoff parameters depending on 16S rRNA sequence analysis method. Using data analyzed with amplicon sequence variant error correction, Piphillin outperformed comparable tools, for instance exhibiting 19% greater balanced accuracy and 54% greater precision compared to PICRUSt2. CONCLUSIONS: Our results demonstrate that raw Illumina sequences should be processed for subsequent Piphillin analysis using amplicon sequence variant error correction (with DADA2 or similar methods) and run using a 99% ID cutoff for Piphillin, while sequences generated on platforms other than Illumina should be processed via OTU clustering (e.g., UPARSE) and run using a 96% ID cutoff for Piphillin. Piphillin is publicly available for academic users (Piphillin server. http://piphillin.secondgenome.com/.).
Assuntos
Metagenômica/métodos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Software , Bases de Dados de Ácidos NucleicosRESUMO
Following the publication of this article [1], the authors reported errors in Figs. 1, 2 and 5. Due to a typesetting error the asterisks denoting significance were missing from the published figures.
RESUMO
OBJECTIVE: To determine whether using 10 mL formula after each breastfeeding before copious maternal milk production affects breastfeeding duration, readmission, and intestinal microbiota through 1 month of age. STUDY DESIGN: In this randomized controlled trial, we enrolled 164 exclusively breastfeeding newborns, 24-72 hours old, whose weight loss was ≥75th percentile for age, and whose mothers had not yet begun mature milk production. Enrolled newborns were assigned randomly to either supplement breastfeeding with early limited formula (ELF), 10 mL of formula after each breastfeeding stopped at the onset of copious maternal milk production (intervention), or to continue exclusive breastfeeding (control). Outcomes assessed through 1 month included breastfeeding duration, readmission, and intestinal microbiota. RESULTS: At 1 week of age, 95.8% of infants receiving ELF and 93.5% of control infants were still breastfeeding (P > .5); readmission occurred for 4 (4.8%) control infants and none of the infants receiving ELF (P = .06). At 1 month of age, 86.5% of infants receiving ELF and 89.7% of control infants were still breastfeeding (P > .5); 54.6% of infants receiving ELF and 65.8% of controls were breastfeeding without formula (P = .18). ELF did not lead to decreased abundance of Lactobacillus or Bifidobacterium and was not associated with expansion of Clostridium. CONCLUSION: In this population of healthy newborns with weight loss ≥75th percentile, ELF did not interfere with breastfeeding at 1 month, breastfeeding without formula at 1 month, or intestinal microbiota. ELF may be an important therapeutic option for newborns with the potential to reduce readmission rates. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02313181.
Assuntos
Aleitamento Materno , Microbioma Gastrointestinal , Fórmulas Infantis , Readmissão do Paciente , Humanos , Recém-Nascido , Leite HumanoRESUMO
Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10(-13); adjusted p= 2.2 x 10(-3)). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 - 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.
Assuntos
Proteínas ADAM/genética , Encéfalo/anormalidades , Fenda Labial/genética , Fissura Palatina/genética , Estudo de Associação Genômica Ampla , Proteínas ADAMTS , Animais , Encéfalo/patologia , Fenda Labial/patologia , Fissura Palatina/patologia , Cães , Mutação da Fase de Leitura , Haplótipos , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
PURPOSE: To evaluate outcomes after intraoperative radiation therapy (IORT) in high-risk neuroblastoma (NB), including local control, overall survival, and toxicity. METHODS AND MATERIALS: This was a single institution retrospective study of 92 pediatric patients with NB treated with IORT from 1995 to 2022. Each IORT application was considered a separate event for a total of 110 sites treated. Local failure was calculated using the cumulative incidence function and survival by Kaplan-Meier method from the day of surgery. RESULTS: All patients had high-risk relapsed or treatment refractory disease. Median age was 6 years (range, 2-34 years). Median follow-up for all patients and surviving patients was 16 months and 4 years, respectively. All patients previously received chemotherapy, 93% had prior external beam radiation therapy to the site of IORT (median dose, 21.6 Gy; range, 10-36 Gy), and 94% had a prior surgery for tumor resection. The median IORT dose was 12 Gy (range, 8-18 Gy) and median area treated was 18 cm2 (range, 2.5-60 cm2). The cumulative incidence of local failure was 23% at 2 years and 29% at 5 years. The overall survival (OS) was 44% at 2 years and 29% at 5 years. Local failure after IORT was associated with worse OS (hazard ratio, 1.74; 95% CI, 1.07-2.84; P = .0267). Toxicity from IORT was rare, with postoperative complications likely related to IORT seen in 7 (8%) patients. CONCLUSIONS: Our study represents the largest, most recent analysis of the efficacy and safety of IORT in patients with relapsed or refractory NB. Less than one-third of patients failed locally at 5 years, and achieving local control affected overall survival. Minimal toxicities directly related to IORT were observed. Overall, IORT is an effective and safe technique to achieve local control in high-risk relapsed or refractory neuroblastoma.
Assuntos
Recidiva Local de Neoplasia , Neuroblastoma , Humanos , Neuroblastoma/radioterapia , Neuroblastoma/mortalidade , Neuroblastoma/cirurgia , Criança , Pré-Escolar , Masculino , Feminino , Adolescente , Estudos Retrospectivos , Adulto , Adulto Jovem , Cuidados Intraoperatórios/métodosRESUMO
Motivation: Microbial metagenomic profiling software and databases are advancing rapidly for development of novel disease biomarkers and therapeutics yet three problems impede analyses: 1) the conflation of "genome assembly" and "strain" in reference databases; 2) difficulty connecting DNA biomarkers to a procurable strain for laboratory experimentation; and 3) absence of a comprehensive and unified strain-resolved reference database for integrating both shotgun metagenomics and 16S rRNA gene data. Results: We demarcated 681,087 strains, the largest collection of its kind, by filtering public data into a knowledge graph of vertices representing contiguous DNA sequences, genome assemblies, strain monikers and bio-resource center (BRC) catalog numbers then adding inter-vertex edges only for synonyms or direct derivatives. Surprisingly, for 10,043 important strains, we found replicate RefSeq genome assemblies obstructing interpretation of database searches. We organized each strain into eight taxonomic ranks with bootstrap confidence inversely correlated with genome assembly contamination. The StrainSelect database is suited for applications where a taxonomic, functional or procurement reference is needed for shotgun or amplicon metagenomics since 636,568 strains have at least one 16S rRNA gene, 245,005 have at least one annotated genome assembly, and 36,671 are procurable from at least one BRC. The database overcomes all three aforementioned problems since it disambiguates strains from assemblies, locates strains at BRCs, and unifies a taxonomic reference for both 16S rRNA and shotgun metagenomics. Availability: The StrainSelect database is available in igraph and tabular vertex-edge formats compatible with Neo4J. Dereplicated MinHash and fasta databases are distributed for sourmash and usearch pipelines at http://strainselect.secondgenome.com. Contact:todd.desantis@gmail.com. Supplementary information: Supplementary data are available online.
RESUMO
Objective: Inflammatory bowel disease (IBD) is a heterogenous disease in which the microbiome has been shown to play an important role. However, the precise homeostatic or pathological functions played by bacteria remain unclear. Most published studies report taxa-disease associations based on single-technology analysis of a single cohort, potentially biasing results to one clinical protocol, cohort, and molecular analysis technology. To begin to address this key question, precise identification of the bacteria implicated in IBD across cohorts is necessary. Methods: We sought to take advantage of the numerous and diverse studies characterizing the microbiome in IBD to develop a multi-technology meta-analysis (MTMA) as a platform for aggregation of independently generated datasets, irrespective of DNA-profiling technique, in order to uncover the consistent microbial modulators of disease. We report the largest strain-level survey of IBD, integrating microbiome profiles from 3,407 samples from 21 datasets spanning 15 cohorts, three of which are presented for the first time in the current study, characterized using three DNA-profiling technologies, mapping all nucleotide data against known, culturable strain reference data. Results: We identify several novel IBD associations with culturable strains that have so far remained elusive, including two genome-sequenced but uncharacterized Lachnospiraceae strains consistently decreased in both the gut luminal and mucosal contents of patients with IBD, and demonstrate that these strains are correlated with inflammation-related pathways that are known mechanisms targeted for treatment. Furthermore, comparative MTMA at the species versus strain level reveals that not all significant strain associations resulted in a corresponding species-level significance and conversely significant species associations are not always re-captured at the strain level. Conclusion: We propose MTMA for uncovering experimentally testable strain-disease associations that, as demonstrated here, are beneficial in discovering mechanisms underpinning microbiome impact on disease or novel targets for therapeutic interventions.
RESUMO
BACKGROUND: Both the gut microbiota and chronic viral infections have profound effects on host immunity, but interactions between these influences have been only superficially explored. Cytomegalovirus (CMV), for example, infects approximately 80% of people globally and drives significant changes in immune cells. Similarly, certain gut-resident bacteria affect T-cell development in mice and nonhuman primates. It is unknown if changes imposed by CMV on the intestinal microbiome contribute to immunologic effects of the infection. RESULTS: We show that rhesus cytomegalovirus (RhCMV) infection is associated with specific differences in gut microbiota composition, including decreased abundance of Firmicutes, and that the extent of microbial change was associated with immunologic changes including the proliferation, differentiation, and cytokine production of CD8+ T cells. Furthermore, RhCMV infection disrupted the relationship between short-chain fatty acid producers and Treg/Th17 balance observed in seronegative animals, showing that some immunologic effects of CMV are due to disruption of previously existing host-microbe relationships. CONCLUSIONS: Gut microbes have an important influence on health and disease. Diet is known to shape the microbiota, but the influence of concomitant chronic viral infections is unclear. We found that CMV influences gut microbiota composition to an extent that is correlated with immunologic changes in the host. Additionally, pre-existing correlations between immunophenotypes and gut microbes can be subverted by CMV infection. Immunologic effects of CMV infection on the host may therefore be mediated by two different mechanisms involving gut microbiota. Video Abstract.
Assuntos
Infecções por Citomegalovirus , Linfócitos T Reguladores , Animais , Linfócitos T CD8-Positivos , Citocinas , Citomegalovirus/genética , Ácidos Graxos Voláteis , Macaca mulatta , CamundongosRESUMO
Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Adolescente , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Organoides/patologiaRESUMO
Breastfeeding is the gold standard for feeding infants because of its long-term benefits to health and development, but most infants in the United States are not exclusively breastfed in the first six months. We enrolled 24 infants who were either exclusively breastfed or supplemented with formula by the age of one month. We collected diet information, stool samples for evaluation of microbiotas by 16S rRNA sequencing, and blood samples for assessment of immune development by flow cytometry from birth to 6 months of age. We further typed the Bifidobacterium strains in stool samples whose 16S rRNA sequencing showed the presence of Bifidobacteriaceae. Supplementation with formula during breastfeeding transiently changed the composition of the gut microbiome, but the impact dissipated by six months of age. For example, Bifidobacterium longum, a bacterial species highly correlated with human milk consumption, was found to be significantly different only at 1 month of age but not at later time points. No immunologic differences were found to be associated with supplementation, including the development of T-cell subsets, B cells, or monocytes. These data suggest that early formula supplementation, given in addition to breast milk, has minimal lasting impact on the gut microbiome or immunity.
Assuntos
Suplementos Nutricionais/microbiologia , Microbioma Gastrointestinal/imunologia , Sistema Imunitário/crescimento & desenvolvimento , Fórmulas Infantis/microbiologia , Fenômenos Fisiológicos da Nutrição do Lactente/imunologia , Aleitamento Materno/métodos , Inquéritos sobre Dietas , Fezes/microbiologia , Feminino , Humanos , Sistema Imunitário/microbiologia , Lactente , Recém-Nascido , Masculino , RNA Ribossômico 16S/isolamento & purificação , Estados UnidosRESUMO
To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzofuranos/uso terapêutico , Carcinoma Hepatocelular/genética , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Naftoquinonas/uso terapêutico , Sulfonamidas/uso terapêuticoRESUMO
Hepatoblastoma is the most common childhood liver cancer. Although survival has improved significantly over the past few decades, there remains a group of children with aggressive disease who do not respond to current treatment regimens. There is a critical need for novel models to study aggressive hepatoblastoma as research to find new treatments is hampered by the small number of laboratory models of the disease. Organoids have emerged as robust models for many diseases, including cancer. We have generated and characterized a novel organoid model of aggressive hepatoblastoma directly from freshly resected patient tumors as a proof of concept for this approach. Hepatoblastoma tumor organoids recapitulate the key elements of patient tumors, including tumor architecture, mutational profile, gene expression patterns, and features of Wnt/ß-catenin signaling that are hallmarks of hepatoblastoma pathophysiology. Tumor organoids were successfully used alongside non-tumor liver organoids from the same patient to perform a drug screen using twelve candidate compounds. One drug, JQ1, demonstrated increased destruction of liver organoids from hepatoblastoma tumor tissue relative to organoids from the adjacent non-tumor liver. Our findings suggest that hepatoblastoma organoids could be used for a variety of applications and have the potential to improve treatment options for the subset of hepatoblastoma patients who do not respond to existing treatments.
RESUMO
Early infant diet has significant impacts on the gut microbiota and developing immune system. We previously showed that breast-fed and formula-fed rhesus macaques develop significantly different gut microbial communities, which in turn are associated with different immune systems in infancy. Breast-fed animals manifested greater T cell activation and proliferation and harbored robust pools of T helper 17 (TH17) cells. These differences were sustained throughout the first year of life. Here we examine groups of juvenile macaques (approximately 3 to 5 y old), which were breast-fed or formula-fed in infancy. We demonstrate that juveniles breast-fed in infancy maintain immunologic differences into the fifth year of life, principally in CD8(+) memory T cell activation. Additionally, long-term correlation networks show that breast-fed animals maintain persistent relationships between immune subsets that are not seen in formula-fed animals. These findings demonstrate that infant feeding practices have continued influence on immunity for up to 3 to 5 y after birth and also reveal mechanisms for microbial modulation of the immune system.
Assuntos
Dieta/métodos , Microbioma Gastrointestinal , Sistema Imunitário/fisiologia , Animais , Animais Recém-Nascidos , Linfócitos T CD8-Positivos , Memória Imunológica , Fórmulas Infantis/administração & dosagem , Macaca mulatta , Leite HumanoRESUMO
Diet has a strong influence on the intestinal microbiota in both humans and animal models. It is well established that microbial colonization is required for normal development of the immune system and that specific microbial constituents prompt the differentiation or expansion of certain immune cell subsets. Nonetheless, it has been unclear how profoundly diet might shape the primate immune system or how durable the influence might be. We show that breast-fed and bottle-fed infant rhesus macaques develop markedly different immune systems, which remain different 6 months after weaning when the animals begin receiving identical diets. In particular, breast-fed infants develop robust populations of memory T cells as well as T helper 17 (TH17) cells within the memory pool, whereas bottle-fed infants do not. These findings may partly explain the variation in human susceptibility to conditions with an immune basis, as well as the variable protection against certain infectious diseases.