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1.
Anal Chim Acta ; 1178: 338551, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482862

RESUMO

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 µm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 µm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis.


Assuntos
Análise de Célula Única , Cromatografia Líquida , Espectrometria de Massas
2.
BMC Bioinformatics ; 22(Suppl 10): 419, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34479487

RESUMO

BACKGROUND: RNA velocity is a novel and powerful concept which enables the inference of dynamical cell state changes from seemingly static single-cell RNA sequencing (scRNA-seq) data. However, accurate estimation of RNA velocity is still a challenging problem, and the underlying kinetic mechanisms of transcriptional and splicing regulations are not fully clear. Moreover, scRNA-seq data tend to be sparse compared with possible cell states, and a given dataset of estimated RNA velocities needs imputation for some cell states not yet covered. RESULTS: We formulate RNA velocity prediction as a supervised learning problem of classification for the first time, where a cell state space is divided into equal-sized segments by directions as classes, and the estimated RNA velocity vectors are considered as ground truth. We propose Velo-Predictor, an ensemble learning pipeline for predicting RNA velocities from scRNA-seq data. We test different models on two real datasets, Velo-Predictor exhibits good performance, especially when XGBoost was used as the base predictor. Parameter analysis and visualization also show that the method is robust and able to make biologically meaningful predictions. CONCLUSION: The accurate result shows that Velo-Predictor can effectively simplify the procedure by learning a predictive model from gene expression data, which could help to construct a continous landscape and give biologists an intuitive picture about the trend of cellular dynamics.


Assuntos
RNA , Análise de Célula Única , Perfilação da Expressão Gênica , Aprendizado de Máquina , RNA/genética , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
3.
Nat Commun ; 12(1): 5127, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493721

RESUMO

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals.


Assuntos
Gatos/genética , Regulação da Expressão Gênica no Desenvolvimento , Pigmentação/genética , Animais , Animais Domésticos , Gatos/crescimento & desenvolvimento , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Genótipo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Mutação , Fenótipo , Análise de Célula Única , Pele/anatomia & histologia , Pele/crescimento & desenvolvimento , Pele/metabolismo , Via de Sinalização Wnt
4.
Nat Commun ; 12(1): 4774, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362886

RESUMO

Biological N2 fixation was key to the expansion of life on early Earth. The N2-fixing microorganisms and the nitrogenase type used in the Proterozoic are unknown, although it has been proposed that the canonical molybdenum-nitrogenase was not used due to low molybdenum availability. We investigate N2 fixation in Lake Cadagno, an analogue system to the sulfidic Proterozoic continental margins, using a combination of biogeochemical, molecular and single cell techniques. In Lake Cadagno, purple sulfur bacteria (PSB) are responsible for high N2 fixation rates, to our knowledge providing the first direct evidence for PSB in situ N2 fixation. Surprisingly, no alternative nitrogenases are detectable, and N2 fixation is exclusively catalyzed by molybdenum-nitrogenase. Our results show that molybdenum-nitrogenase is functional at low molybdenum conditions in situ and that in contrast to previous beliefs, PSB may have driven N2 fixation in the Proterozoic ocean.


Assuntos
Chromatiaceae/metabolismo , Molibdênio/metabolismo , Fixação de Nitrogênio , Nitrogênio/metabolismo , Biomassa , Ciclo do Carbono , Dióxido de Carbono , Tamanho Celular , Chromatiaceae/genética , Metagenoma , Modelos Teóricos , Nitrogenase/metabolismo , Oceanos e Mares , Análise de Célula Única
5.
Nat Commun ; 12(1): 4763, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362918

RESUMO

The comparison of gene regulatory networks between diseased versus healthy individuals or between two different treatments is an important scientific problem. Here, we propose sc-compReg as a method for the comparative analysis of gene expression regulatory networks between two conditions using single cell gene expression (scRNA-seq) and single cell chromatin accessibility data (scATAC-seq). Our software, sc-compReg, can be used as a stand-alone package that provides joint clustering and embedding of the cells from both scRNA-seq and scATAC-seq, and the construction of differential regulatory networks across two conditions. We apply the method to compare the gene regulatory networks of an individual with chronic lymphocytic leukemia (CLL) versus a healthy control. The analysis reveals a tumor-specific B cell subpopulation in the CLL patient and identifies TOX2 as a potential regulator of this subpopulation.


Assuntos
Redes Reguladoras de Genes , Leucemia Linfocítica Crônica de Células B/genética , Análise de Célula Única/métodos , Linfócitos B , Cromatina , Regulação Neoplásica da Expressão Gênica , Proteínas HMGB , Humanos , RNA Citoplasmático Pequeno , Software
6.
Nat Commun ; 12(1): 4797, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376651

RESUMO

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.


Assuntos
Suturas Cranianas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Acrocefalossindactilia/embriologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Suturas Cranianas/citologia , Suturas Cranianas/embriologia , Dura-Máter/citologia , Dura-Máter/embriologia , Dura-Máter/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq/métodos , Crânio/citologia , Crânio/embriologia , Crânio/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo
8.
Nat Commun ; 12(1): 4940, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400634

RESUMO

Understanding the genetic and epigenetic programs that control differentiation during development is a fundamental challenge, with broad impacts across biology and medicine. Measurement technologies like single-cell RNA-sequencing and CRISPR-based lineage tracing have opened new windows on these processes, through computational trajectory inference and lineage reconstruction. While these two mathematical problems are deeply related, methods for trajectory inference are not typically designed to leverage information from lineage tracing and vice versa. Here, we present LineageOT, a unified framework for lineage tracing and trajectory inference. Specifically, we leverage mathematical tools from graphical models and optimal transport to reconstruct developmental trajectories from time courses with snapshots of both cell states and lineages. We find that lineage data helps disentangle complex state transitions with increased accuracy using fewer measured time points. Moreover, integrating lineage tracing with trajectory inference in this way could enable accurate reconstruction of developmental pathways that are impossible to recover with state-based methods alone.


Assuntos
Diferenciação Celular , Linhagem da Célula/genética , Animais , Caenorhabditis elegans , Expressão Gênica , Humanos , Análise de Sequência de RNA , Análise de Célula Única
9.
Elife ; 102021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34424199

RESUMO

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns-including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups-and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.


Assuntos
COVID-19/imunologia , Infecções Comunitárias Adquiridas/imunologia , Influenza Humana/imunologia , Análise de Célula Única , Adulto , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade
10.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360605

RESUMO

Piezo1/2 are mechanosensitive calcium-permeable channels that can be activated by various modes of membrane deformation. The identification of the small molecule Yoda1, a synthetic Piezo1 agonist, revealed the possibility of chemical activation of the channel. Stimulating effects of Yoda1 on Piezo1 have been mainly documented using over-expressing cellular systems or channel proteins incorporated in artificial lipid bilayers. However, the activating effect of Yoda1 on native Piezo1 channels in the plasma membrane of living cells remains generally undefined, despite the increasing number of studies in which the agonist is utilized as a functional tool to reveal the contribution of Piezo1 to cellular reactions. In the current study, we used the human myeloid leukemia K562 cell line as a suitable model to examine chemically induced Piezo1 activity with the use of the patch-clamp technique in various specific modes. The functional expression of Piezo1 in leukemia cells was evidenced using a combinative approach, including single channel patch-clamp measurements. Utilizing our established single-current whole-cell assay on K562 cells, we have shown, for the first time, the selective real-time chemical activation of endogenously expressed Piezo1. Extracellular application of 0.5-1 µM Yoda1 effectively stimulated single Piezo1 currents in the cell membrane.


Assuntos
Membrana Celular/metabolismo , Canais Iônicos/efeitos dos fármacos , Leucemia/tratamento farmacológico , Mecanotransdução Celular , Pirazinas/farmacologia , Análise de Célula Única/métodos , Tiadiazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Humanos , Canais Iônicos/agonistas , Canais Iônicos/metabolismo , Leucemia/metabolismo , Leucemia/patologia
11.
Nat Commun ; 12(1): 4821, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376672

RESUMO

Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease.


Assuntos
Genoma Viral/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Adulto , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Evolução Clonal/genética , Células Clonais/metabolismo , Células Clonais/patologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Mutação , RNA-Seq/métodos , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T/genética , Fator de Transcrição STAT3/genética
12.
Analyst ; 146(17): 5307-5315, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34351328

RESUMO

Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.


Assuntos
Fenômenos Biológicos , Proteínas Proto-Oncogênicas c-akt , Digitonina , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Análise de Célula Única
13.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445499

RESUMO

Xenoestrogens and phytoestrogens are referred to as "foreign estrogens" that are produced outside of the human body and have been shown to exert estrogen-like activity. Xenoestrogens are synthetic industrial chemicals, whereas phytoestrogens are chemicals present in the plant. Considering that these environmental estrogen mimics potentially promote hormone-related cancers, an understanding of how they interact with estrogenic pathways in human cells is crucial to resolve their possible impacts in cancer. Here, we conducted an extensive literature evaluation on the origins of these chemicals, emerging research techniques, updated molecular mechanisms, and ongoing clinical studies of estrogen mimics in human cancers. In this review, we describe new applications of patient-derived xenograft (PDX) models and single-cell RNA sequencing (scRNA-seq) techniques in shaping the current knowledge. At the molecular and cellular levels, we provide comprehensive and up-to-date insights into the mechanism of xenoestrogens and phytoestrogens in modulating the hallmarks of cancer. At the systemic level, we bring the emerging concept of window of susceptibility (WOS) into focus. WOS is the critical timing during the female lifespan that includes the prenatal, pubertal, pregnancy, and menopausal transition periods, during which the mammary glands are more sensitive to environmental exposures. Lastly, we reviewed 18 clinical trials on the application of phytoestrogens in the prevention or treatment of different cancers, conducted from 2002 to the present, and provide evidence-based perspectives on the clinical applications of phytoestrogens in cancers. Further research with carefully thought-through concepts and advanced methods on environmental estrogens will help to improve understanding for the identification of environmental influences, as well as provide novel mechanisms to guide the development of prevention and therapeutic approaches for human cancers.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/prevenção & controle , Fitoestrógenos/uso terapêutico , Análise de Célula Única/métodos , Animais , Biomarcadores Tumorais/genética , Ensaios Clínicos como Assunto , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias/genética , Fitoestrógenos/farmacologia , Análise de Sequência de RNA , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445507

RESUMO

Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, we compare standard and emerging single-cell techniques and their relevance for investigating human diseases. Finally, we describe emerging methodologies that investigate centromeric chromatin specification and neocentromere formation.


Assuntos
Centrômero/metabolismo , Cromatina/metabolismo , Análise de Célula Única/métodos , Montagem e Desmontagem da Cromatina , Epigênese Genética , Humanos
15.
Oxid Med Cell Longev ; 2021: 5633514, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34457116

RESUMO

This study sought to perform integrative analysis of the immune/methylation/autophagy landscape on breast cancer prognosis and single-cell genotypes. Breast Cancer Recurrence Risk Score (BCRRS) and Breast Cancer Prognostic Risk Score (BCPRS) were determined based on 6 prognostic IMAAGs obtained from the TCGA-BRCA cohort. BCRRS and BCPRS, respectively, were used to construct a risk prediction model of overall survival and progression-free survival. Predictive capacity of the model was evaluated using clinical data. Analysis showed that BCRRS is associated with a high risk of stroke. In addition, PPI and drug-ceRNA networks based on differences in BCPRS were constructed. Single cells were genotyped through integrated scRNA-seq of the TNBC samples based on clustering results of BCPRS-related genes. The findings of this study show the potential regulatory effects of IMAAGs on breast cancer tumor microenvironment. High AUCs of 0.856 and 0.842 were obtained for the OS and PFS prognostic models, respectively. scRNA-seq analysis showed high expression levels of adipocytes and adipose tissue macrophages (ATMs) in high BCPRS clusters. Moreover, analysis of ligand-receptor interactions and potential regulatory mechanisms were performed. The LINC00276&MALAT1/miR-206/FZD4-Wnt7b pathway was also identified which may be useful in future research on targets against breast cancer metastasis and recurrence. Neural network-based deep learning models using BCPRS-related genes showed that these genes can be used to map the tumor microenvironment. In summary, analysis of IMAAGs, BCPRS, and BCRRS provides information on the breast cancer microenvironment at both the macro- and microlevels and provides a basis for development of personalized treatment therapy.


Assuntos
Autofagia , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Metilação de DNA , Análise de Célula Única/métodos , Acidente Vascular Cerebral/patologia , Microambiente Tumoral/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Terapia Combinada , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genótipo , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Prognóstico , Medição de Risco/métodos , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/terapia , Taxa de Sobrevida , Transcriptoma
16.
Clin Immunol ; 230: 108820, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365017

RESUMO

BACKGROUND: Peanut oral immunotherapy (POIT) has provided desensitization to peanut allergic individuals. Limited immunological evaluation exists during the first 24-weeks of POIT. OBJECTIVE: Regulatory T-cells (Tregs) are antigen induced immunosuppressive T-cells important in establishing tolerance. Delineation of early immunologic changes contributing to the development of peanut desensitization would help clarify the mechanism of action in POIT. We performed single-cell RNA sequencing (scRNAseq) on Tregs in pediatric subjects undergoing POIT during the first 24-weeks of therapy to evaluate early immunological changes induced by POIT. METHODS: PBMC samples from peanut allergic subjects between 5 and 12 years of age enrolled in a Phase 1/2a POIT study were collected and analyzed at 0, 6, and 24-weeks after POIT initiation and samples were compared to healthy non-peanut allergic controls. Tregs were enriched from PBMCs and scRNAseq analysis performed. Cell Ranger 3.1.0 (10× Genomics) was utilized to identify cell clusters and differentially expressed genes, and results were analyzed with Seurat suite version 3.0.0. RESULTS: Gene analysis revealed 10 major clusters corresponding to different cell types observed to change during POIT when compared to the healthy, non-peanut-allergic state. scRNAseq analysis of Tregs revealed strong CD3G expression correlating with gdTregs. scRNAseq analysis of gdTregs revealed dynamic changes occurring within the first 6-weeks of treatment and cell frequencies of naïve and memory gdTregs at 24-weeks of treatment reducing to levels similar to healthy controls. Analysis of transcriptomic cell identity analysis using SingleR showed gene expression in gdTregs similar to healthy control after 24-weeks of POIT treatment. scRNAseq analysis revealed alterations in gene expression for memory and naïve gdTregs during this timeframe. Specifically, expression of OX40R (TNFRSF4), GITR (TNFRSF18), TGFB1, CTLA4, ISG20, CD69 were upregulated in memory gdTregs compared to naive gdTregs by 24-weeks of POIT, while IL7R and SELL were downregulated in memory gdTregs compared to naïve gdTregs. CONCLUSIONS: There are specific expression profiles of peripheral naïve and mature gdTreg cells in peanut allergic patients undergoing POIT in the first 24-weeks of treatment implicating pathways involved in maintenance of immune homeostasis. gdTreg cells may contribute to the tolerogenic effect of POIT within the first 24-weeks of POIT treatment. These findings suggest that gdTregs cells may be an early marker of desensitization in subjects undergoing POIT.


Assuntos
Arachis/imunologia , Dessensibilização Imunológica/métodos , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T , Hipersensibilidade a Amendoim/terapia , Linfócitos T Reguladores/imunologia , Administração Oral , Alérgenos/administração & dosagem , Criança , Pré-Escolar , Humanos , Memória Imunológica , Família Multigênica , Hipersensibilidade a Amendoim/genética , Hipersensibilidade a Amendoim/imunologia , RNA-Seq , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Análise de Célula Única , Fatores de Tempo , Transcriptoma
17.
BMC Bioinformatics ; 22(1): 416, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34461827

RESUMO

BACKGROUND: Genetic heterogeneity of a cancer tumor that develops during clonal evolution is one of the reasons for cancer treatment failure, by increasing the chance of drug resistance. Clones are cell populations with different genotypes, resulting from differences in somatic mutations that occur and accumulate during cancer development. An appropriate approach for identifying clones is determining the variant allele frequency of mutations that occurred in the tumor. Although bulk sequencing data can be used to provide that information, the frequencies are not informative enough for identifying different clones with the same prevalence and their evolutionary relationships. On the other hand, single-cell sequencing data provides valuable information about branching events in the evolution of a cancerous tumor. However, the temporal order of mutations may be determined with ambiguities using only single-cell data, while variant allele frequencies from bulk sequencing data can provide beneficial information for inferring the temporal order of mutations with fewer ambiguities. RESULT: In this study, a new method called Conifer (ClONal tree Inference For hEterogeneity of tumoR) is proposed which combines aggregated variant allele frequency from bulk sequencing data with branching event information from single-cell sequencing data to more accurately identify clones and their evolutionary relationships. It is proven that the accuracy of clone identification and clonal tree inference is increased by using Conifer compared to other existing methods on various sets of simulated data. In addition, it is discussed that the evolutionary tree provided by Conifer on real cancer data sets is highly consistent with information in both bulk and single-cell data. CONCLUSIONS: In this study, we have provided an accurate and robust method to identify clones of tumor heterogeneity and their evolutionary history by combining single-cell and bulk sequencing data.


Assuntos
Neoplasias , Traqueófitas , Evolução Clonal , Genótipo , Humanos , Mutação , Neoplasias/genética , Análise de Célula Única
18.
Medicine (Baltimore) ; 100(31): e26775, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34397824

RESUMO

ABSTRACT: Rhabdomyosarcoma is the most common soft tissue sarcoma in children, and embryonal rhabdomyosarcoma is the most typical type of rhabdomyosarcoma. The heterogeneity, etiology, and origin of embryonal rhabdomyosarcoma remain unknown.After obtaining the gene expression data of every cell in the tumor tissue by single-cell RNA sequencing, we used the Seurat package in R studio for quality control, analysis, and exploration of the data. All cells are divided into tumor cells and non-tumor cells, and we chose tumor cells by marker genes. Then, we repeated the process to cluster the tumor cells and divided the subgroups by their differentially expressed genes and gene ontology/Kyoto Encyclopedia of Genes and Genomes analysis. Additionally, Monocle 2 was used for pseudo-time analysis to obtain the evolution trajectory of cells in tumor tissues.Tumor cells were divided into 5 subgroups according to their functions, which were characterized by high proliferation, sensing and adaptation to oxygen availability, enhanced epigenetic modification, enhanced nucleoside phosphonic acid metabolism, and ossification. Evolution trajectory of cells in tumor tissues is obtained.We used pseudo-time analysis to distinguish between mesenchymal stem cells and fibroblasts, proved that embryonal rhabdomyosarcoma in the pelvic originated from skeletal muscle progenitor cells, showed the evolutionary trajectory of embryonal rhabdomyosarcoma, and improved the method of evaluating the degree of malignancy of embryonal rhabdomyosarcoma.


Assuntos
Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/patologia , Análise de Célula Única/métodos , Expressão Gênica/genética , Humanos , Pelve/anormalidades , Pelve/diagnóstico por imagem , Análise de Célula Única/estatística & dados numéricos
19.
Talanta ; 234: 122671, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364472

RESUMO

Traditional cell biology researches on cell populations by their origin, tissue, morphology, and secretions. Because of the heterogeneity of cells, research at the single-cell level can obtain more accurate and comprehensive information that reflects the physiological state and process of the cell, increasing the significance of single-cell analysis. The application of single-cell analysis is faced with the problem of contaminated or damaged cells caused by cell sample transportation. Reversible encapsulation of a single cell can protect cells from the external environment and open the encapsulation shell to release cells, thus preserving cell integrity and improving extraction efficiency of analytes. Meanwhile, microfluidic single cell analysis (MSCA) exhibits integration, miniaturization, and high throughput, which can considerably improve the efficiency of single-cell analysis. The researches on single-cell reversible encapsulation materials, single-cell analysis methods, and the MSCA integration platform are analyzed and summarized in this review. The problems of single-cell viability, network of single-cell signal, and simultaneous detection of multiple biotoxins in food based on single-cell are proposed for future research.


Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Análise de Célula Única
20.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
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