RESUMEN
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.
Asunto(s)
Colitis Ulcerosa/patología , Colon/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Bestrofinas/metabolismo , Antígenos CD8/metabolismo , Estudios de Casos y Controles , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Colon/patología , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Interleucina-17/metabolismo , Masculino , Persona de Mediana Edad , Factores de Riesgo , Linfocitos T/citología , Linfocitos T/metabolismo , Trombospondinas/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
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.
Asunto(s)
Proteínas , ARN , Proteínas/química , ARN/química , Técnicas In Vitro , MultiómicaRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Cromatina/genética , Aprendizaje Profundo , Regulación de la Expresión Génica , Análisis de la Célula Individual/métodos , Programas Informáticos , Transcriptoma , Animales , Cromatina/química , Cromatina/metabolismo , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , RNA-Seq , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
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.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Ratones , Bulbo Olfatorio/citología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Análisis de Matrices TisularesRESUMEN
There is limited evidence showing that elevated arterial blood pressure in surgical patients may be associated with increased perioperative risk; however, cardiovascular instability frequently occurs during anesthesia. The most commonly used anesthetic agents, both intravenous and inhalation ones, produce a decrease in arterial blood pressure. Magnesium, acting as a natural calcium-channel blocker, induces direct and indirect vasodilatation, thus playing a role in the treatment of arterial hypertension. In this research, we assessed the effects of magnesium sulfate on cardiovascular stability in patients undergoing diverse planned surgical procedures (abdominal, orthopedic, urology) under general balanced anesthesia, who were diagnosed with arterial hypertension grade 1 and 2. The research encompassed 100 patients of both sexes, aged from 20 to 65. Immediately before induction of anesthesia with propofol, the patients in the experimental group (50 study subjects) received 30 mg/ kg bolus dose and magnesium sulfate infusion at 10 mg/kg/h, whereas the subjects in the control group (50 patients) were administered normal saline. Anesthesia was achieved and maintained with sevoflurane, fentanyl and rocuronium. The hemodynamic variables of mean arterial pressure and heart rate were measured every five minutes, starting immediately before magnesium infusion. Statistical analysis of the categorized values of mean arterial pressure and heart rate revealed a statistically significant between-group difference at 60th and 90th minute of anesthesia. In conclusion, magnesium sulfate as an adjuvant to anesthesia in patients with arterial hypertension reduces hemodynamic changes during anesthesia.
Asunto(s)
Anestesia/métodos , Anestésicos Combinados/administración & dosificación , Hemodinámica/efectos de los fármacos , Hipertensión/fisiopatología , Sulfato de Magnesio/administración & dosificación , Adulto , Anestesiología , Presión Sanguínea , Método Doble Ciego , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Quirúrgicos OperativosRESUMEN
DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.
Asunto(s)
Anticuerpos/química , Proteínas Sanguíneas/análisis , ADN/química , Proteómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunoensayo/métodos , Inmunoconjugados/química , Separación Inmunomagnética/métodos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.
Asunto(s)
Anticuerpos Inmovilizados/química , Biotecnología/métodos , Separación Inmunomagnética/métodos , Nanotecnología/métodos , Biotina , Colorantes FluorescentesRESUMEN
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.
RESUMEN
Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.
Asunto(s)
Metilación de ADN , Genética Forense , Líquidos Corporales/química , Epigenómica , Marcadores Genéticos , HumanosRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Artritis Reumatoide , Transcriptoma , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Humanos , Membrana Sinovial/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antibacterianos/administración & dosificación , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/epidemiología , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Pruebas de Sensibilidad Microbiana , Vigilancia de la Población , Estudios Retrospectivos , Serbia/epidemiologíaRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expresión Génica , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Microglía/metabolismo , Microglía/patología , Neuronas Motoras/patología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Neuroglía/metabolismo , Neuroglía/patología , Cambios Post Mortem , Análisis Espacio-Temporal , Médula Espinal/patología , TranscriptomaRESUMEN
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.
Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Bulbo Olfatorio/metabolismo , ARN/genética , Análisis de Matrices Tisulares/métodos , Transcriptoma , Animales , Código de Barras del ADN Taxonómico/instrumentación , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Microtomía , Bulbo Olfatorio/ultraestructura , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Microextracción en Fase Sólida/métodos , Coloración y Etiquetado/métodos , Análisis de Matrices Tisulares/instrumentación , Fijación del Tejido/métodosAsunto(s)
Venas Hepáticas , Vena Cava Superior Izquierda Persistente , Humanos , Venas Hepáticas/cirugía , Vena Cava Superior/diagnóstico por imagen , Vena Cava Superior/cirugía , Vena Cava Superior/anomalías , Esternotomía , Atrios Cardíacos/diagnóstico por imagen , Atrios Cardíacos/cirugía , DrenajeRESUMEN
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.
Asunto(s)
Adenocarcinoma/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Transcriptoma/genética , Adenocarcinoma/patología , Adenocarcinoma/cirugía , Biología Computacional , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Masculino , Próstata/citología , Próstata/patología , Próstata/cirugía , Prostatectomía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , ARN Mensajero/genética , Células del Estroma/patología , Microambiente Tumoral/genéticaRESUMEN
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.