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1.
bioRxiv ; 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38712088

RESUMO

Tissue structure and molecular circuitry in the colon can be profoundly impacted by systemic age-related effects, but many of the underlying molecular cues remain unclear. Here, we built a cellular and spatial atlas of the colon across three anatomical regions and 11 age groups, encompassing ~1,500 mouse gut tissues profiled by spatial transcriptomics and ~400,000 single nucleus RNA-seq profiles. We developed a new computational framework, cSplotch, which learns a hierarchical Bayesian model of spatially resolved cellular expression associated with age, tissue region, and sex, by leveraging histological features to share information across tissue samples and data modalities. Using this model, we identified cellular and molecular gradients along the adult colonic tract and across the main crypt axis, and multicellular programs associated with aging in the large intestine. Our multi-modal framework for the investigation of cell and tissue organization can aid in the understanding of cellular roles in tissue-level pathology.

2.
Nat Biotechnol ; 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37985876

RESUMO

Mucosal and barrier tissues, such as the gut, lung or skin, are composed of a complex network of cells and microbes forming a tight niche that prevents pathogen colonization and supports host-microbiome symbiosis. Characterizing these networks at high molecular and cellular resolution is crucial for understanding homeostasis and disease. Here we present spatial host-microbiome sequencing (SHM-seq), an all-sequencing-based approach that captures tissue histology, polyadenylated RNAs and bacterial 16S sequences directly from a tissue by modifying spatially barcoded glass surfaces to enable simultaneous capture of host transcripts and hypervariable regions of the 16S bacterial ribosomal RNA. We applied our approach to the mouse gut as a model system, used a deep learning approach for data mapping and detected spatial niches defined by cellular composition and microbial geography. We show that subpopulations of gut cells express specific gene programs in different microenvironments characteristic of regional commensal bacteria and impact host-bacteria interactions. SHM-seq should enhance the study of native host-microbe interactions in health and disease.

3.
J Med Biochem ; 42(3): 469-475, 2023 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-37790204

RESUMO

Background: Surgical stress and pain result in activation of hypothalamus-pituitary-adrenal axis. The aim of this study was to establish the effects of postoperative pain and various modalities of analgesic administration on salivary and serum cortisol levels, as well as to establish the validity of salivary cortisol as a stress indicator in surgical patients. Methods: A randomized controlled trial involved 60 patients scheduled for elective abdominal aortic aneurysm surgery. Patients were randomly divided into two groups depending on the model of postoperative analgesia. The first group (MI - morphine intermittently) included patients given morphine doses 0.1 mg/kg/6h s.c. intermittently. The second group (MPCA - morphine patient-controlled analgesia) included patients who received morphine via the PCA system - intravenous administration of morphine adjusted to a dose of 1 mg per shot and a lockout interval of 6 minutes.

6.
Nat Methods ; 20(3): 424-431, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36864197

RESUMO

As spatially resolved multiplex profiling of RNA and proteins becomes more prominent, it is increasingly important to understand the statistical power available to test specific hypotheses when designing and interpreting such experiments. Ideally, it would be possible to create an oracle that predicts sampling requirements for generalized spatial experiments. However, the unknown number of relevant spatial features and the complexity of spatial data analysis make this challenging. Here, we enumerate multiple parameters of interest that should be considered in the design of a properly powered spatial omics study. We introduce a method for tunable in silico tissue (IST) generation and use it with spatial profiling data sets to construct an exploratory computational framework for spatial power analysis. Finally, we demonstrate that our framework can be applied across diverse spatial data modalities and tissues of interest. While we demonstrate ISTs in the context of spatial power analysis, these simulated tissues have other potential use cases, including spatial method benchmarking and optimization.


Assuntos
Proteínas , RNA , Proteínas/química , RNA/química , Técnicas In Vitro , Multiômica
7.
Commun Biol ; 5(1): 129, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35149753

RESUMO

The inflamed rheumatic joint is a highly heterogeneous and complex tissue with dynamic recruitment and expansion of multiple cell types that interact in multifaceted ways within a localized area. Rheumatoid arthritis synovium has primarily been studied either by immunostaining or by molecular profiling after tissue homogenization. Here, we use Spatial Transcriptomics, where tissue-resident RNA is spatially labeled in situ with barcodes in a transcriptome-wide fashion, to study local tissue interactions at the site of chronic synovial inflammation. We report comprehensive spatial RNA-Seq data coupled to cell type-specific localization patterns at and around organized structures of infiltrating leukocyte cells in the synovium. Combining morphological features and high-throughput spatially resolved transcriptomics may be able to provide higher statistical power and more insights into monitoring disease severity and treatment-specific responses in seropositive and seronegative rheumatoid arthritis.


Assuntos
Artrite Reumatoide , Transcriptoma , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Humanos , Membrana Sinovial/metabolismo
8.
Nat Methods ; 18(11): 1352-1362, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34711971

RESUMO

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.


Assuntos
Encéfalo/metabolismo , Cromatina/genética , Aprendizado Profundo , Regulação da Expressão Gênica , Análise de Célula Única/métodos , Software , Transcriptoma , Animais , Cromatina/química , Cromatina/metabolismo , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Sequências Reguladoras de Ácido Nucleico
9.
J Med Biochem ; 39(3): 346-354, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-33269023

RESUMO

BACKGROUND: The goal of this study was to assess the oxidative stress status through the values of antioxidant defense parameters: superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and total antioxidant status (TAS), as well as cardiovascular risk factors (total cholesterol, LDL-cholesterol, VLDL-cholesterol, non-HDL-cholesterol and triglycerides), anthropometric parameters (Body mass index-BMI, waist circumference-WC, hipp circumferemce-HC, waist-to-hipp ratio-WHR and inflammatory markers (high sensitive C-reactive protein) in a group of obese adolescents. METHODS: A total of 238 students of both sexes, age of 22.32 ± 1.85 yr. were included in the study. According to the values of BMI lower and higher than 25 kg/m2 and WC higher and lower than 94 cm (males)/80 cm (females) the tested group of students was divided into 2 subgroups: Group 1 (increased risk for CVD) and Group 2 (lower risk for CVD). RESULTS: Significantly reduced SOD and GPx with increased GR, TAS, inflammatory and lipoprotein parameters were obtained in Group 1 compared to Group 2. Significant positive association of hsCRP (OR:1.41; 95%CI 1.08-1.83; P=0.007), TAS (OR:827.2; 95%CI 19.27-35498; P=0.007) and GR (OR:1.13; 95%CI 1.05-1.21; P=0.002) and negative association of GPx (OR:0.97; 95%CI 0.94-1.003; P=0.043) and HDL-cholesterol (OR:0.41; 95%CI 0.176-0.963; P=0.0014) with cardiovascular risk factors were found in obese students. According to the ROC analysis GR>44.8 U/L, TAS>1.35 mmol/L, hsCRP>1.71 mg/L and HDL-cholesterol <1.13 mmol/L have sufficient predictive ability for cardiovascular disease in obese students. CONCLUSIONS: Significant association of antioxidant defense parameters with anthropometric, lipid and inflammatory markers in obese students with increased cardiovascular risk suggest that screening of these parameters is necessary and highly recommended.

10.
J Chemother ; 32(6): 294-303, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32321359

RESUMO

A surveillance study was performed in an intensive care unit in the largest tertiary health care center in Vojvodina, Serbia from 2014 to 2018. Antibiotic prescription data were collated in the WHO anatomical therapeutic chemical (ATC)/defined daily dose (DDD) format, while antibiotic resistance was expressed as incidence density adjusted for total inpatient-days. Individual trends were determined by linear regression, while possible associations between antibiotic prescription and resistance were evaluated using cross-correlation analysis. An overall decrease in antibiotic utilization was observed. The prescription rates of piperacillin-tazobactam increased significantly, while consumption of 3rd and 4th generation cephalosporins and fluoroquinolones decreased. There were rising incidence densities of doripenem resistant Acinetobacter spp., piperacillin-tazobactam resistant Pseudomonas aeruginosa and carbapenem and colistin resistant Klebsiella pneumoniae. These results can serve as a basis for the development of antimicrobial stewardship strategies in the current setting.


Assuntos
Antibacterianos/administração & dosagem , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Testes de Sensibilidade Microbiana , Vigilância da População , Estudos Retrospectivos , Sérvia/epidemiologia
11.
Nat Methods ; 16(10): 987-990, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501547

RESUMO

Spatial and molecular characteristics determine tissue function, yet high-resolution methods to capture both concurrently are lacking. Here, we developed high-definition spatial transcriptomics, which captures RNA from histological tissue sections on a dense, spatially barcoded bead array. Each experiment recovers several hundred thousand transcript-coupled spatial barcodes at 2-µm resolution, as demonstrated in mouse brain and primary breast cancer. This opens the way to high-resolution spatial analysis of cells and tissues.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Bulbo Olfatório/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Análise Serial de Tecidos
12.
Cell ; 178(3): 714-730.e22, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348891

RESUMO

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.


Assuntos
Colite Ulcerativa/patologia , Colo/metabolismo , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Bestrofinas/metabolismo , Antígenos CD8/metabolismo , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Colo/patologia , Enterócitos/citologia , Enterócitos/metabolismo , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Interleucina-17/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Linfócitos T/citologia , Linfócitos T/metabolismo , Trombospondinas/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
13.
Science ; 364(6435): 89-93, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948552

RESUMO

Paralysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify pathway dynamics, distinguish regional differences between microglia and astrocyte populations at early time points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.


Assuntos
Esclerose Lateral Amiotrófica/genética , Expressão Gênica , Neurônios Motores/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Microglia/metabolismo , Microglia/patologia , Neurônios Motores/patologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Neuroglia/metabolismo , Neuroglia/patologia , Mudanças Depois da Morte , Análise Espaço-Temporal , Medula Espinal/patologia , Transcriptoma
14.
Nat Protoc ; 13(11): 2501-2534, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30353172

RESUMO

Spatial resolution of gene expression enables gene expression events to be pinpointed to a specific location in biological tissue. Spatially resolved gene expression in tissue sections is traditionally analyzed using immunohistochemistry (IHC) or in situ hybridization (ISH). These technologies are invaluable tools for pathologists and molecular biologists; however, their throughput is limited to the analysis of only a few genes at a time. Recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data in bulk. Spatial Transcriptomics combines the benefits of traditional spatially resolved technologies with the massive throughput of RNA-seq. Here, we present a protocol describing how to apply the Spatial Transcriptomics technology to mammalian tissue. This protocol combines histological staining and spatially resolved RNA-seq data from intact tissue sections. Once suitable tissue-specific conditions have been established, library construction and sequencing can be completed in ~5-6 d. Data processing takes a few hours, with the exact timing dependent on the sequencing depth. Our method requires no special instruments and can be performed in any laboratory with access to a cryostat, microscope and next-generation sequencing.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Bulbo Olfatório/metabolismo , RNA/genética , Análise Serial de Tecidos/métodos , Transcriptoma , Animais , Código de Barras de DNA Taxonômico/instrumentação , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Microtomia , Bulbo Olfatório/ultraestrutura , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microextração em Fase Sólida/métodos , Coloração e Rotulagem/métodos , Análise Serial de Tecidos/instrumentação , Fixação de Tecidos/métodos
15.
Nat Commun ; 9(1): 2419, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925878

RESUMO

Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.


Assuntos
Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Transcriptoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Biologia Computacional , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Masculino , Próstata/citologia , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , RNA Mensageiro/genética , Células Estromais/patologia , Microambiente Tumoral/genética
16.
Sci Rep ; 7(1): 12941, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-29021611

RESUMO

Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.


Assuntos
Biomarcadores/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade
17.
Nat Plants ; 3: 17061, 2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28481330

RESUMO

Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.


Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Genes de Plantas , Picea/genética , Populus/genética , Estudos de Viabilidade , Reprodutibilidade dos Testes
19.
Nat Commun ; 7: 13182, 2016 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-27739429

RESUMO

Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes.


Assuntos
Biotecnologia/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Animais , Células Cultivadas , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Células MCF-7 , Camundongos , Células NIH 3T3
20.
Science ; 353(6294): 78-82, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27365449

RESUMO

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Encéfalo/metabolismo , Neoplasias da Mama/metabolismo , DNA Complementar/biossíntese , Feminino , Humanos , Camundongos , Especificidade de Órgãos , RNA Mensageiro/metabolismo
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