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Phosphate-solubilizing bacteria (PSB) are crucial for enhancing phosphorus bioavailability and regulating phosphorus transformation processes. However, the in situ phosphorus-solubilizing activity and the link between phenotypes and genotypes for PSB remain unidentified. Here we employed single-cell Raman spectroscopy combined with heavy water to discern and quantify soil active PSB. Our results reveal that PSB abundance and in situ activity differed significantly between soil types and fertilization treatments. Inorganic fertilizer input was the key driver for active PSB distribution. Targeted single-cell sorting and metagenomic sequencing of active PSB uncovered several low-abundance genera that are easily overlooked within bulk soil microbiota. We elucidate the underlying functional genes and metabolic pathway, and the interplay between phosphorus and carbon cycling involved in high phosphorus solubilization activity. Our study provides a single-cell approach to exploring PSB from native environments, enabling the development of a microbial solution for the efficient agronomic use of phosphorus and mitigating the phosphorus crisis.
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INTRODUCTION: Reflux bile acids are thought to promote EAC, but the role of systemic bile acids is unknown. This study aimed to assess associations between systemic bile acids and stages of Barrett's esophagus (BE) progression. METHODS: Subjects with and without BE were enrolled in this multi-center cross-sectional study. Targeted serum bile acid profiling was performed, and a subset of subjects completed a validated food frequency questionnaire. RNA sequencing was performed on BE or gastric cardia tissue to assess bile acid associations with gene expression. RESULTS: 141 subjects were enrolled with serum bile acids profiled (49 non-BE; 92 BE: 44 no dysplasia, 25 indefinite/low grade dysplasia, 23 high grade dysplasia/EAC). Lower Healthy Eating Index score, older age, higher body mass index, and no proton pump inhibitor use were associated with increased levels of multiple bile acids. Global bile acid pools were distinct between non-BE and stages of BE neoplasia (p=0.004). Increasing cholic acid was associated with high grade dysplasia/EAC compared to non-BE, even after adjusting for EAC risk factors (aOR 2.03, 95% CI 1.11-3.71) as was the combination of unconjugated primary bile acids (aOR 1.81, 95% CI 1.04-3.13). High cholic acid levels were associated with tissue gene expression changes including increased DNA replication and reduced lymphocyte differentiation genes. DISCUSSION: Alterations in serum bile acids are independently associated with advanced neoplasia in BE and may contribute to neoplastic progression. Future studies should explore associated gut microbiome changes, pro-neoplastic effects of bile acids, and whether these bile acids, particularly cholic acid, represent potential biomarkers or viable therapeutic targets for advanced neoplasia in BE.
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The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features ⢠Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq ⢠Targeted strategy for enriching low-frequency stromal cells.
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Transfer learning provides a powerful tool for incorporating data from related studies into a target study of interest. In epidemiology and medical studies, the classification of a target disease could borrow information across other related diseases and populations. In this work, we consider transfer learning for high-dimensional generalized linear models (GLMs). A novel algorithm, TransHDGLM, that integrates data from the target study and the source studies is proposed. Minimax rate of convergence for estimation is established and the proposed estimator is shown to be rate-optimal. Statistical inference for the target regression coefficients is also studied. Asymptotic normality for a debiased estimator is established, which can be used for constructing coordinate-wise confidence intervals of the regression coefficients. Numerical studies show significant improvement in estimation and inference accuracy over GLMs that only use the target data. The proposed methods are applied to a real data study concerning the classification of colorectal cancer using gut microbiomes, and are shown to enhance the classification accuracy in comparison to methods that only use the target data.
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Plastic pollution represents a critical threat to soil ecosystems and even humans, as plastics can serve as a habitat for breeding and refuging pathogenic microorganisms against stresses. However, evaluating the health risk of plastispheres is difficult due to the lack of risk factors and quantification model. Here, DNA sequencing, single-cell Raman-D2O labeling, and transformation assay were used to quantify key risk factors of plastisphere, including pathogen abundance, phenotypic resistance to various stresses (antibiotic and pesticide), and ability to acquire antibiotic resistance genes. A Bayesian network model was newly introduced to integrate these three factors and infer their causal relationships. Using this model, the risk of pathogen in the plastisphere is found to be nearly 3 magnitudes higher than that in free-living state. Furthermore, this model exhibits robustness for risk prediction, even in the absence of one factor. Our framework offers a novel and practical approach to assessing the health risk of plastispheres, contributing to the management of plastic-related threats to human health.
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Teorema de Bayes , Bactérias/genética , Bactérias/isolamento & purificação , Fenótipo , Microbiologia do Solo , Humanos , Antibacterianos/farmacologiaRESUMO
Purpose: To evaluate the ability of swept-source optical coherence tomography angiography (SS-OCTA) to assess lid margin vascularity. Methods: This prospective, cross-sectional trial enrolled 125 participants, including 15 control subjects and 110 meibomian gland dysfunction (MGD) patients. Lid margin blood flow density (LMBFD) was obtained using SS-OCTA. LMBFD was assessed for repeatability in 54 of 125 participants and for reproducibility in 23 of 125 participants. The efficacy of LMBFD was validated in the 125 participants, who were divided into mild (n = 46), moderate (n = 42), and severe groups (n = 37) according to the lid margin vascularity severity shown in the slit-lamp photographs. Correlations between LMBFD and MG-related parameters, such as ocular surface disease index (OSDI), fluorescein tear break-up time (FTBUT), cornea fluorescein staining (CFS), lid margin score (LMS), and meibomian gland expressibility (ME), were analyzed in all 125 participants. Results: Repeatability and reproducibility coefficients were satisfactorily high in the scan mode with a scan area of 6 mm × 6 mm (intraclass correlation coefficient [ICC] repeatability = 0.905; ICC reproducibility = 0.986) and a scan area of 9 mm × 9 mm (ICC repeatability = 0.888; ICC reproducibility = 0.988). The LMBFD gradually increased in the mild, moderate, and severe groups (P < 0.001). LMBFD was significant correlated with OSDI (r = 0.290, P = 0.001), FTBUT (r = -0.195, P = 0.030), CFS (r = 0.352, P < 0.001), ME (r = 0.191, P = 0.033), and LMS (r = 0.370, P < 0.001). Conclusions: LMBFD may be a noninvasive, repeatable, reproducible, and efficient index for the quantitative evaluation of eyelid margin vascularity in the future. Translational Relevance: We demonstrated that SS-OCTA has the potential to evaluate the eyelid margin vascularity in MGD patients and guide future treatment strategies in clinics.
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Pálpebras , Tomografia de Coerência Óptica , Humanos , Tomografia de Coerência Óptica/métodos , Masculino , Feminino , Estudos Prospectivos , Estudos Transversais , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto , Pálpebras/irrigação sanguínea , Pálpebras/diagnóstico por imagem , Idoso , Disfunção da Glândula Tarsal/diagnóstico por imagem , Fluxo Sanguíneo Regional , Angiografia/métodos , Glândulas Tarsais/diagnóstico por imagemRESUMO
Large-scale microbiome studies are progressively utilizing multiomics designs, which include the collection of microbiome samples together with host genomics and metabolomics data. Despite the increasing number of data sources, there remains a bottleneck in understanding the relationships between different data modalities due to the limited number of statistical and computational methods for analyzing such data. Furthermore, little is known about the portability of general methods to the metagenomic setting and few specialized techniques have been developed. In this review, we summarize and implement some of the commonly used methods. We apply these methods to real data sets where shotgun metagenomic sequencing and metabolomics data are available for microbiome multiomics data integration analysis. We compare results across methods, highlight strengths and limitations of each, and discuss areas where statistical and computational innovation is needed.
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Biologia Computacional , Genômica , Metabolômica , Metagenômica , Microbiota , Metabolômica/métodos , Microbiota/genética , Biologia Computacional/métodos , Metagenômica/métodos , Genômica/métodos , HumanosRESUMO
Xylem serves as a conduit linking soil to the aboveground plant parts and facilitating the upward movement of microbes into leaves and fruits. Despite this potential, the composition of the xylem microbiome and its associated risks, including antibiotic resistance, are understudied. Here, we cultivated tomatoes and analyzed their xylem sap to assess the microbiome and antibiotic resistance profiles following treatment with sewage sludge. Our findings show that xylem microbes primarily originate from soil, albeit with reduced diversity in comparison to those of their soil microbiomes. Using single-cell Raman spectroscopy coupled with D2O labeling, we detected significantly higher metabolic activity in xylem microbes than in rhizosphere soil, with 87% of xylem microbes active compared to just 36% in the soil. Additionally, xylem was pinpointed as a reservoir for antibiotic resistance genes (ARGs), with their abundance being 2.4-6.9 times higher than in rhizosphere soil. Sludge addition dramatically increased the abundance of ARGs in xylem and also increased their mobility and host pathogenicity. Xylem represents a distinct ecological niche for microbes and is a significant reservoir for ARGs. These results could be used to manage the resistome in crops and improve food safety.
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Resistência Microbiana a Medicamentos , Esgotos , Solanum lycopersicum , Xilema , Solanum lycopersicum/microbiologia , Solanum lycopersicum/genética , Esgotos/microbiologia , Resistência Microbiana a Medicamentos/genética , Microbiologia do Solo , Rizosfera , MicrobiotaRESUMO
BACKGROUND: Cellular immunotherapy, represented by the chimeric antigen receptor T cell (CAR-T), has exhibited high response rates, durable remission, and safety in vitro and in clinical trials. Unfortunately, anti-CD19 CAR-T (CART-19) treatment alone is prone to relapse and has a particularly poor prognosis in relapsed/refractory (r/r) B-ALL patients. To date, addressing or reducing relapse remains one of the research priorities to achieve broad clinical application. METHODS: We manufactured second generation CART-19 cells and validated their efficacy and safety in vitro and in vivo. Through co-culture of Nalm-6 cells with short-term cultured CART-19 cells, CD19-negative Nalm-6 cells were detected by flow cytometry, and further investigation of the relapsed cells and their resistance mechanisms was evaluated in vitro. RESULTS: In this study, we demonstrated that CART-19 cells had enhanced and specific antileukemic activities, and the survival of B-ALL mouse models after CART-19 treatment was significantly prolonged. We then shortened the culture time and applied the serum-free culture to expand CAR-T cells, followed by co-culturing CART-19 cells with Nalm-6 cells. Surprisingly, we observed the proliferation of CD19-negative Nalm-6 cells around 28 days. Identification of potential resistance mechanisms showed that the relapsed cells express truncated CD19 proteins with decreased levels and, more importantly, CAR expression was detected on the relapsed cell surface, which may ultimately keep them antigen-negative. Furthermore, it was validated that CART-22 and tandem CART-22/19 cells could effectively kill the relapsed cells, but neither could completely eradicate them. CONCLUSIONS: We successfully generated CART-19 cells and obtained a CD19-negative refractory relapsed B-ALL cell line, providing new insights into the underlying mechanisms of resistance and a new in vitro model for the treatment of r/r B-ALL patients with low antigen density.
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Antígenos CD19 , Receptores de Antígenos Quiméricos , Antígenos CD19/metabolismo , Antígenos CD19/imunologia , Animais , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Resistencia a Medicamentos Antineoplásicos , Camundongos , Técnicas de Cocultura , Ensaios Antitumorais Modelo de Xenoenxerto , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologiaRESUMO
Microbially-driven soil formation process is an emerging technology for the ecological rehabilitation of alkaline tailings. However, the dominant microorganisms and their specific roles in soil formation processes remain unknown. Herein, a 1-year field-scale experiment was applied to demonstrate the effect of nitrogen input on the structure and function of the microbiome in alkaline bauxite residue. Results showed that the contents of nutrient components were increased with Penicillium oxalicum (P. oxalicum) incorporation, as indicated by the increasing of carbon and nitrogen mineralization and enzyme metabolic efficiency. Specifically, the increasing enzyme metabolic efficiency was associated with nitrogen input, which shaped the microbial nutrient acquisition strategy. Subsequently, we evidenced that P. oxalicum played a significant role in shaping the assemblages of core bacterial taxa and influencing ecological functioning through intra- and cross-kingdom network analysis. Furthermore, a recruitment experiment indicated that nitrogen enhanced the enrichment of core microbiota (Nitrosomonas, Bacillus, Pseudomonas, and Saccharomyces) and may provide benefits to fungal community bio-diversity and microbial network stability. Collectively, these results demonstrated nitrogen-based coexistence patterns among P. oxalicum and microbiome and revealed P. oxalicum-mediated nutrient dynamics and ecophysiological adaptations in alkaline microhabitats. It will aid in promoting soil formation and ecological rehabilitation of bauxite residue. ENVIRONMENT IMPLICATION: Bauxite residue is a highly alkaline solid waste generated during the Bayer process for producing alumina. Attempting to transform bauxite residue into a stable soil-like substrate using low-cost microbial resources is a highly promising engineering. However, the dominant microorganisms and their specific roles in soil formation processes remain unknown. In this study, we evidenced the nitrogen-based coexistence patterns among Penicillium oxalicum and microbiome and revealed Penicillium oxalicum-mediated nutrient dynamics and ecophysiological adaptations in alkaline microhabitats. This study can improve the understanding of core microbes' assemblies that affect the microbiome physiological traits in soil formation processes.
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Óxido de Alumínio , Bactérias , Microbiota , Nitrogênio , Penicillium , Microbiologia do Solo , Penicillium/metabolismo , Penicillium/crescimento & desenvolvimento , Nitrogênio/metabolismo , Óxido de Alumínio/química , Bactérias/metabolismo , Bactérias/crescimento & desenvolvimento , Solo/químicaRESUMO
BACKGROUND & AIMS: Crohn's disease is associated with alterations in the gut microbiome and metabolome described as dysbiosis. We characterized the microbial and metabolic consequences of ileal resection, the most common Crohn's disease surgery. METHODS: Patients with and without intestinal resection were identified from the Diet to Induce Remission in Crohn's Disease and Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease studies. Stool samples were analyzed with shotgun metagenomics sequencing. Fecal butyrate was measured with 1H nuclear magnetic resonance spectroscopy. Fecal bile acids and plasma 7α-hydroxy-4-cholesten-3-one (C4) was measured with mass spectrometry. RESULTS: Intestinal resection was associated with reduced alpha diversity and altered beta diversity with increased Proteobacteria and reduced Bacteroidetes and Firmicutes. Surgery was associated with higher representation of genes in the KEGG pathway for ABC transporters and reduction in genes related to bacterial metabolism. Surgery was associated with reduced concentration of the But gene but this did not translate to reduced fecal butyrate concentration. Surgery was associated with decreased abundance of bai operon genes, with increased plasma C4 concentration, increased primary bile acids and reduced secondary bile acids, including isoLCA. Additionally, Egerthella lenta, Adlercreutzia equalofaciens, and Gordonibacter pamelaeae were lower in abundance among patients with prior surgery in both cohorts. CONCLUSIONS: In 2 different populations, prior surgery in Crohn's disease is associated with altered fecal microbiome. Patients who had undergone ileal resection had reduction in the potentially beneficial bacteria E lenta and related actinobacteria and secondary bile acids, including isoLCA, suggesting that these could be biomarkers of patients at higher risk for disease progression.
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Doença de Crohn , Disbiose , Fezes , Microbioma Gastrointestinal , Metaboloma , Humanos , Doença de Crohn/microbiologia , Doença de Crohn/cirurgia , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Feminino , Masculino , Adulto , Estudos Prospectivos , Fezes/microbiologia , Disbiose/microbiologia , Pessoa de Meia-Idade , Ácidos e Sais Biliares/metabolismo , Butiratos/metabolismo , Metagenômica/métodos , Colestenonas/metabolismo , Íleo/microbiologia , Íleo/cirurgia , Íleo/metabolismo , Íleo/patologia , Adulto Jovem , Bactérias/isolamento & purificação , Bactérias/classificação , Bactérias/metabolismo , Bactérias/genéticaRESUMO
Aerobic anoxygenic phototrophic bacteria (AAPB) contribute profoundly to the global carbon cycle. However, most AAPB in marine environments are uncultured and at low abundance, hampering the recognition of their functions and molecular mechanisms. In this study, we developed a new culture-independent method to identify and sort AAPB using single-cell Raman/fluorescence spectroscopy. Characteristic Raman and fluorescent bands specific to bacteriochlorophyll a (Bchl a) in AAPB were determined by comparing multiple known AAPB with non-AAPB isolates. Using these spectroscopic biomarkers, AAPB in coastal seawater, pelagic seawater, and hydrothermal sediment samples were screened, sorted, and sequenced. 16S rRNA gene analysis and functional gene annotations of sorted cells revealed novel AAPB members and functional genes, including one species belonging to the genus Sphingomonas, two genera affiliated to classes Betaproteobacteria and Gammaproteobacteria, and function genes bchCDIX, pucC2, and pufL related to Bchl a biosynthesis and photosynthetic reaction center assembly. Metagenome-assembled genomes (MAGs) of sorted cells from pelagic seawater and deep-sea hydrothermal sediment belonged to Erythrobacter sanguineus that was considered as an AAPB and genus Sphingomonas, respectively. Moreover, multiple photosynthesis-related genes were annotated in both MAGs, and comparative genomic analysis revealed several exclusive genes involved in amino acid and inorganic ion metabolism and transport. This study employed a new single-cell spectroscopy method to detect AAPB, not only broadening the taxonomic and genetic contents of AAPB in marine environments but also revealing their genetic mechanisms at the single-genomic level.
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Metagenômica , Água do Mar , Metagenômica/métodos , Água do Mar/microbiologia , RNA Ribossômico 16S/genética , Análise Espectral Raman , Filogenia , Análise de Célula ÚnicaRESUMO
INTRODUCTION: There are limited longitudinal data on the impact of chronic therapy on the natural history of eosinophilic esophagitis (EoE), a chronic allergic disease of the esophagus. The purpose of this study was to evaluate if patients with well-controlled EoE were less likely to develop fibrostenotic complications. METHODS: Subjects were identified from a database of pediatric patients with EoE at the Children's Hospital of Philadelphia started in 2000. Patients were then searched in adult medical records to identify patients who transitioned care. All office visits, emergency department visits, and endoscopic, histologic, and imaging reports were reviewed for the primary outcome of strictures and the secondary outcomes of food impactions and dysphagia. Cox proportional hazard regression was performed for outcomes. RESULTS: One hundred five patients were identified with the mean follow-up of 11.4 ± 4.9 years. 52.3% (n = 55) had a period of histologic disease control defined as ≥2 consecutive endoscopies with histologic remission. These patients were less likely to develop strictures compared with patients who did not have a period of histologic control (HR 0.232; 95% CI 0.084-0.64, P = 0.005). Patients who were diagnosed at younger ages were less likely to develop strictures. Presentation with dysphagia or impaction was associated with higher rate of stricture development. DISCUSSION: In this cohort study with > 10 years of follow-up, children with EoE with a period of histologic disease control and diagnosed at younger ages were less likely to develop esophageal strictures. While this suggests histologic remission is associated with reduction of remodeling complications, additional prospective data with long-term follow-up are needed.
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BACKGROUND: Immunotherapy has emerged as a potent clinical approach for cancer treatment, but only subsets of cancer patients can benefit from it. Targeting lactate metabolism (LM) in tumor cells as a method to potentiate anti-tumor immune responses represents a promising therapeutic strategy. METHODS: Public single-cell RNA-Seq (scRNA-seq) cohorts collected from patients who received immunotherapy were systematically gathered and scrutinized to delineate the association between LM and the immunotherapy response. A novel LM-related signature (LM.SIG) was formulated through an extensive examination of 40 pan-cancer scRNA-seq cohorts. Then, multiple machine learning (ML) algorithms were employed to validate the capacity of LM.SIG for immunotherapy response prediction and survival prognostication based on 8 immunotherapy transcriptomic cohorts and 30 The Cancer Genome Atlas (TCGA) pan-cancer datasets. Moreover, potential targets for immunotherapy were identified based on 17 CRISPR datasets and validated via in vivo and in vitro experiments. RESULTS: The assessment of LM was confirmed to possess a substantial relationship with immunotherapy resistance in 2 immunotherapy scRNA-seq cohorts. Based on large-scale pan-cancer data, there exists a notably adverse correlation between LM.SIG and anti-tumor immunity as well as imbalance infiltration of immune cells, whereas a positive association was observed between LM.SIG and pro-tumorigenic signaling. Utilizing this signature, the ML model predicted immunotherapy response and prognosis with an AUC of 0.73/0.80 in validation sets and 0.70/0.87 in testing sets respectively. Notably, LM.SIG exhibited superior predictive performance across various cancers compared to published signatures. Subsequently, CRISPR screening identified LDHA as a pan-cancer biomarker for estimating immunotherapy response and survival probability which was further validated using immunohistochemistry (IHC) and spatial transcriptomics (ST) datasets. Furthermore, experiments demonstrated that LDHA deficiency in pancreatic cancer elevated the CD8+ T cell antitumor immunity and improved macrophage antitumoral polarization, which in turn enhanced the efficacy of immunotherapy. CONCLUSIONS: We unveiled the tight correlation between LM and resistance to immunotherapy and further established the pan-cancer LM.SIG, holds the potential to emerge as a competitive instrument for the selection of patients suitable for immunotherapy.
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Imunoterapia , Neoplasias , Humanos , Imunoterapia/métodos , Prognóstico , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/mortalidade , Neoplasias/metabolismo , Neoplasias/genética , Ácido Láctico/metabolismo , Camundongos , Animais , FemininoRESUMO
OBJECTIVE: This study aimed to investigate the clinical significance and risk factors of postoperative pancreatic fistula (POPF) after post-pancreatectomy acute pancreatitis (PPAP) in patients who underwent pancreaticoduodenectomy (PD). SUMMARY BACKGROUND DATA: PPAP has been recognized as a critical factor in the pathophysiology of POPF after PD. METHODS: A total of 817 consecutive patients who underwent elective PD between January 2020 and June 2022 were included. PPAP and POPF were defined in accordance with the International Study Group for Pancreatic Surgery (ISGPS) definitions. Multivariate logistic analyses were performed to investigate the risk factors for POPF. Comparisons between PPAP-associated POPF and non-PPAP-associated POPF were made to further characterize this intriguing complication. RESULTS: Overall, 159 (19.5%) patients developed POPF after PD, of which 73 (45.9%) occurred following PPAP, and the remaining 86 (54.1%) had non-PPAP-associated POPF. Patients with PPAP-associated POPF experienced significantly higher morbidity than patients without POPF. Multivariate analyses revealed distinct risk factors for each POPF type. For PPAP-associated POPF, independent risk factors included estimated blood loss >200 mL (OR 1.93), MPD ≤3 cm (OR 2.88), and soft pancreatic texture (OR 2.01), largely overlapping with FRS (Fistula Risk Score) elements. On the other hand, non-PPAP-associated POPF was associated with age >65 years (OR 1.95), male (OR 2.10), and MPD ≤3 cm (OR 2.57). Notably, among patients with PPAP, the incidence of POPF consistently hovered around 50% regardless of the FRS stratification. CONCLUSIONS: PPAP-associated POPF presents as a distinct pathophysiology in the development of POPF after PD, potentially opening doors for future prevention strategies targeting the early postoperative period.
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ABSTRACT: Natural killer (NK) cells represent the cytotoxic member within the innate lymphoid cell (ILC) family that are important against viral infections and cancer. Although the NK cell emergence from hematopoietic stem and progenitor cells through multiple intermediate stages and the underlying regulatory gene network has been extensively studied in mice, this process is not well characterized in humans. Here, using a temporal in vitro model to reconstruct the developmental trajectory of NK lineage, we identified an ILC-restricted oligopotent stage 3a CD34-CD117+CD161+CD45RA+CD56- progenitor population, that exclusively gave rise to CD56-expressing ILCs in vitro. We also further investigated a previously nonappreciated heterogeneity within the CD56+CD94-NKp44+ subset, phenotypically equivalent to stage 3b population containing both group-1 ILC and RORγt+ ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1hi S3b and CD161hiCD117hi ILC3 populations distinctively differed in their expression of effector molecules, cytokine secretion, and cytotoxic activity. Furthermore, analysis of lineage output using DNA-barcode tracing across these stages supported a close developmental relationship between S3b-NK and S4-NK (CD56+CD94+) cells, whereas distant to the ILC3 subset. Cross-referencing gene signatures of culture-derived NK cells and other noncytotoxic ILCs with publicly available data sets validated that these in vitro stages highly resemble transcriptional profiles of respective in vivo ILC counterparts. Finally, by integrating RNA velocity and gene network analysis through single-cell regulatory network inference and clustering we unravel a network of coordinated and highly dynamic regulons driving the cytotoxic NK cell program, as a guide map for future studies on NK cell regulation.
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Células Matadoras Naturais , Análise de Célula Única , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Análise de Célula Única/métodos , Linhagem da Célula , Imunidade Inata , Diferenciação CelularRESUMO
Dysbiosis is associated with pediatric and adult-onset inflammatory bowel disease (IBD), but the role of dysbiosis and the microbiome in very early onset IBD (VEO-IBD) has not yet been described. Here, we aimed to demonstrate the impact of age and inflammation on microbial community structure using shotgun metagenomic sequencing in children with VEO-IBD, pediatric-onset IBD, and age-matched pediatric healthy controls (HC) observed longitudinally over the course of 8 weeks. We found disease-related differences in alpha and beta diversity between HC and children with IBD or VEO-IBD. Using a healthy microbial maturity index modeled from HC across the age range to characterize their gut microbiota, we found that children with pediatric-onset IBD and VEO-IBD had lower maturity than their age-matched HC groups, suggesting a disease effect on the microbial community. In addition, patients with pediatric IBD had significantly lower maturity than those with VEO-IBD, who had more heterogeneity at the youngest ages, highlighting differences in these two cohorts that were not captured in standard comparisons of alpha and beta diversity. These results demonstrate that young age and inflammation independently impact microbial community structure. However, the effect is not additive in the youngest patients, likely because of the heterogeneous and dynamic stool microbiome in this population.
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Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Microbiota , Adulto , Humanos , Criança , Lactente , Disbiose , Doenças Inflamatórias Intestinais/epidemiologia , InflamaçãoRESUMO
Epigenetic changes may fill a critical gap in our understanding of kidney disease development, as they not only reflect metabolic changes but are also preserved and transmitted during cell division. We conducted a genome-wide cytosine methylation analysis of 399 human kidney samples, along with single-nuclear open chromatin analysis on over 60,000 cells from 14 subjects, including controls, and diabetes and hypertension attributed chronic kidney disease (CKD) patients. We identified and validated differentially methylated positions associated with disease states, and discovered that nearly 30% of these alterations were influenced by underlying genetic variations, including variants known to be associated with kidney disease in genome-wide association studies. We also identified regions showing both methylation and open chromatin changes. These changes in methylation and open chromatin significantly associated gene expression changes, most notably those playing role in metabolism and expressed in proximal tubules. Our study further demonstrated that methylation risk scores (MRS) can improve disease state annotation and prediction of kidney disease development. Collectively, our results suggest a causal relationship between epigenetic changes and kidney disease pathogenesis, thereby providing potential pathways for the development of novel risk stratification methods.