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Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.
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Adenosina Trifosfatases , Replicação do DNA , Instabilidade Genômica , Proteostase , Humanos , Adenosina Trifosfatases/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Células HEK293 , Proteínas de Ciclo Celular/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genéticaRESUMO
Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.
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COVID-19/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Animais , Humanos , Camundongos , Células NIH 3T3 , RNA Viral/metabolismo , SARS-CoV-2/metabolismoRESUMO
BACKGROUND: Pediatric acute transverse myelitis (ATM) accounts for 20-30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease. METHODS: We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response. RESULTS: We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis. CONCLUSIONS: Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.
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Astrócitos , Mielite Transversa , Humanos , Mielite Transversa/imunologia , Animais , Feminino , Astrócitos/metabolismo , Astrócitos/imunologia , Criança , Camundongos , Masculino , Adolescente , Plasmócitos/imunologia , Plasmócitos/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/sangue , Camundongos Endogâmicos C57BL , Células Cultivadas , Pré-Escolar , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Medula Espinal/metabolismo , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Single-cell RNA-sequencing (scRNA-seq) is being used extensively to measure the mRNA expression of individual cells from deconstructed tissues, organs and even entire organisms to generate cell atlas references, leading to discoveries of novel cell types and deeper insight into biological trajectories. These massive datasets are usually collected from many samples using different scRNA-seq technology platforms, including the popular SMART-Seq2 (SS2) and 10X platforms. Inherent heterogeneities between platforms, tissues and other batch effects make scRNA-seq data difficult to compare and integrate, especially in large-scale cell atlas efforts; yet, accurate integration is essential for gaining deeper insights into cell biology. We present FIRM, a re-scaling algorithm which accounts for the effects of cell type compositions, and achieve accurate integration of scRNA-seq datasets across multiple tissue types, platforms and experimental batches. Compared with existing state-of-the-art integration methods, FIRM provides accurate mixing of shared cell type identities and superior preservation of original structure without overcorrection, generating robust integrated datasets for downstream exploration and analysis. FIRM is also a facile way to transfer cell type labels and annotations from one dataset to another, making it a reliable and versatile tool for scRNA-seq analysis, especially for cell atlas data integration.
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Perfilação da Expressão Gênica , Análise de Célula Única , Perfilação da Expressão Gênica/métodos , RNA , RNA Mensageiro , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
INTRODUCTION: People with HIV (PWH) have increased risk of atherosclerotic cardiovascular disease (ASCVD) compared to non-PWH, but the reasons for this increased risk remain elusive. We investigated the change in ASCVD risk scores over 4-years to identify clinical factors associated with change in risk scores or high risk scores. METHODS: We conducted a preliminary study using retrospective analysis of PWH, between 40-75 years old, seen at the Evelyn Jordan Center with at least two routine HIV visits. We collected clinical and demographic data and calculated the ASCVD risk scores using the Pooled Cohort Equation. Exploratory analyses examined change in risk score categories over time. Final adjusted analysis examined factors associated with change in continuous risk scores over time. RESULTS: Our sample included 187 PWH, 166 were Black/African American and 79 were female. We found no significant change in ASCVD risk score over time. The risk score was significantly higher in PWH with hepatitis C (7.34%; 95% CI 2.59, 12.09; p=0.003) and trended higher in those with dual hepatitis B/C and hepatitis B compared to those without hepatitis (p=0.07). CONCLUSION: We found that ASCVD risk did not change over a 4-year period among predominantly Black young PWH, but infection with hepatitis C and dual hepatitis B/C were associated with higher ASCVD risk scores. Our findings illustrate the need for further longitudinal studies evaluating change in CVD risk and investigating viral hepatitis as an added potential contributor to increased CVD risk in high-risk, vulnerable populations.
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WashU Epigenome Browser (https://epigenomegateway.wustl.edu/browser/) is a web-based genomic data exploration tool that provides visualization, integration, and analysis of epigenomic datasets. The newly renovated user interface and functions have enabled researchers to engage with the browser and genomic data more efficiently and effectively since 2018. Here, we introduce a new integrated panel design in the browser that allows users to interact with 1D (genomic features), 2D (such as Hi-C), 3D (genome structure), and 4D (time series) data in a single web page. The browser can display three-dimensional chromatin structures with the 3D viewer module. The 4D tracks, called 'Dynamic' tracks, animatedly display time-series data, allowing for a more striking visual impact to identify the gene or genomic region candidates as a function of time. Genomic data, such as annotation features, numerical values, and chromatin interaction data can all be viewed in the dynamic track mode. Imaging data from microscopy experiments can also be displayed in the browser. In addition to software development, we continue to service and expand the data hubs we host for large consortia including 4DN, Roadmap Epigenomics, TaRGET and ENCODE, among others. Our growing user/developer community developed additional track types as plugins, such as qBed and dynseq tracks, which extend the utility of the browser. The browser serves as a foundation for additional genomics platforms including the WashU Virus Genome Browser (for COVID-19 research) and the Comparative Genome Browser. The WashU Epigenome Browser can also be accessed freely through Amazon Web Services at https://epigenomegateway.org/.
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Bases de Dados Genéticas , Epigenoma , Navegador , Humanos , COVID-19/genética , Genoma Humano , Internet , SoftwareRESUMO
BACKGROUND: Grafting is a horticultural practice used widely across woody perennial crop species to fuse together the root and shoot system of two distinct genotypes, the rootstock and the scion, combining beneficial traits from both. In grapevine, grafting is used in nearly 80% of all commercial vines to optimize fruit quality, regulate vine vigor, and enhance biotic and abiotic stress-tolerance. Rootstocks have been shown to modulate elemental composition, metabolomic profiles, and the shape of leaves in the scion, among other traits. However, it is currently unclear how rootstock genotypes influence shoot system gene expression as previous work has reported complex and often contradictory findings. RESULTS: In the present study, we examine the influence of grafting on scion gene expression in leaves and reproductive tissues of grapevines growing under field conditions for three years. We show that the influence from the rootstock genotype is highly tissue and time dependent, manifesting only in leaves, primarily during a single year of our three-year study. Further, the degree of rootstock influence on scion gene expression is driven by interactions with the local environment. CONCLUSIONS: Our results demonstrate that the role of rootstock genotype in modulating scion gene expression is not a consistent, unchanging effect, but rather an effect that varies over time in relation to local environmental conditions.
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Interação Gene-Ambiente , Raízes de Plantas , Raízes de Plantas/metabolismo , Folhas de Planta/genética , Genótipo , Expressão GênicaRESUMO
The dissolution of anthropogenic carbon dioxide (CO2 ) in seawater has altered its carbonate chemistry in the process of ocean acidification (OA). OA affects the viability of marine species. In particular, calcifying organisms and their early planktonic larval stages are considered vulnerable. These organisms often utilize energy reserves for metabolism rather than growth and calcification as supported by bulk RNA-sequencing (RNA-seq) experiments. Yet, transcriptomic profiling of a bulk sample reflects the average gene expression of the population, neglecting the variations between individuals, which forms the basis for natural selection. Here, we used single-embryo RNA-seq on larval sea urchin Heliocidaris crassispina, which is a commercially and ecologically valuable species in East Asia, to document gene expression changes to OA at an individual and family level. Three paternal half-sibs groups were fertilized and exposed to 3 pH conditions (ambient pH 8.0, 7.7 and 7.4) for 12 h prior to sequencing and oxygen consumption assay. The resulting transcriptomic profile of all embryos can be distinguished into four clusters, with differences in gene expressions that govern biomineralization, cell differentiation and patterning, as well as metabolism. While these responses were influenced by pH conditions, the male identities also had an effect. Specifically, a regression model and goodness of fit tests indicated a significant interaction between sire and pH on the probability of embryo membership in different clusters of gene expression. The single-embryo RNA-seq approach is promising in climate stressor research because not only does it highlight potential impacts before phenotypic changes were observed, but it also highlights variations between individuals and lineages, thus enabling a better determination of evolutionary potential.
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Ouriços-do-Mar , Água do Mar , Humanos , Animais , Masculino , Água do Mar/química , Concentração de Íons de Hidrogênio , Ouriços-do-Mar/genética , Perfilação da Expressão Gênica , Larva/fisiologia , Transcriptoma/genética , Dióxido de Carbono/química , Oceanos e MaresRESUMO
Individual mammalian cells exhibit large variability in cellular volume, even with the same absolute DNA content, and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single-molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single-cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, most likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S phase. Our results provide a framework for quantitatively understanding the relationships among DNA content, cell size, and gene expression variability in single cells.
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Dosagem de Genes , Hibridização in Situ Fluorescente/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcrição Gênica , Animais , Caenorhabditis elegans/genética , Células Cultivadas , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Fase SRESUMO
BACKGROUND: Tinea capitis, atopic dermatitis and allergic rhinitis are the most common disorders endured by prepubescent children. Dermatophyte infections have been linked to allergic disorders, such as increased sensitivity to dermatophytes in patients with atopic dermatitis. OBJECTIVES: To explore the correlation between tinea capitis and allergic diseases in children and to analyse their risk factors. METHODS: This study monitored epidemiological changes in childhood tinea capitis and risk factors for whom with allergic disease in a single centre in three consecutive five-year intervals by reviewing clinical data and multivariate logistic data analysis. RESULTS: Between 2007 and 2022, there were 127 children patients with tinea capitis, the mean age was 4.83 years, and the male-to-female ratio was 1.76:1. Zoophilic Microsporum canis and Trichophyton mentagrophytes were the most prevalent pathogens, and the proportions remained relatively constant every 5 years. There were 34 (26.8%) children with tinea capitis complicated with allergic disease, among them 14 children with atopic dermatitis/eczema, 13 with allergic rhinitis, 8 urticaria, 6 food allergies and 1 allergic asthma. Male, kerion, zoophilic species infections and animal contact history were prevalent features in allergic disease combined with tinea capitis. Patients with tinea capitis plus allergic disease mostly had a family history with similar complications. CONCLUSION: M. canis and T. mentagrophytes were the most prevalent pathogens of tinea capitis in the last 15 years; atopic dermatitis/eczema and allergic rhinitis were the most frequently associated allergic diseases. Male, kerion, zoophilic pathogen and animal contact history are risk factors.
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Dermatite Atópica , Eczema , Rinite Alérgica , Tinha do Couro Cabeludo , Animais , Masculino , Feminino , Tinha do Couro Cabeludo/epidemiologia , Microsporum , Fatores de Risco , TrichophytonRESUMO
BACKGROUND: Cell-free DNA (cfDNA) is emerging as a biomarker for sepsis. Previous studies have been focused mainly on identifying blood infections or simply quantifying cfDNA. We propose that by characterizing multifaceted unexplored components, cfDNA could be more informative for assessing this complex syndrome. METHODS: We explored multiple aspects of cfDNA in septic and nonseptic intensive care unit (ICU) patients by metagenomic sequencing, with longitudinal measurement and integrative assessment of plasma cfDNA quantity, human cfDNA fragmentation patterns, infecting pathogens, and overall microbial composition. RESULTS: Septic patients had significantly increased cfDNA quantity and altered human cfDNA fragmentation pattern. Moreover, human cfDNA fragments appeared to comprise information about cellular oxidative stress and could indicate disease severity. Metagenomic sequencing was more sensitive than blood culture in detecting bacterial infections and allowed for simultaneous detection of viral pathogens. We found differences in microbial composition between septic and nonseptic patients and between survivors and nonsurvivors by 28-day mortality, both on the first day of ICU admission and across the study period. By integrating all the information into a machine learning model, we achieved improved performance in identifying sepsis and prediction of clinical outcome for ICU patients with areas under the curve of 0.992 (95% CI 0.969-1.000) and 0.802 (95% CI 0.605-0.999), respectively. CONCLUSIONS: We were able to diagnose sepsis and predict mortality as soon as the first day of ICU admission by integrating multifaceted cfDNA information obtained in a single metagenomic assay; this approach could provide important advantages for clinical management and for improving outcomes in ICU patients.
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Ácidos Nucleicos Livres , Sepse , Biomarcadores , Humanos , Unidades de Terapia Intensiva , Prognóstico , Sepse/diagnóstico , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Smoking is a leading cause of cardiovascular disease (CVD), particularly coronary heart disease (CHD). However, quitting smoking may prevent secondary CVD events in people already diagnosed with CHD. OBJECTIVES: To examine the impact of smoking cessation on death from CVD and major adverse cardiovascular events (MACE), in people with incident CHD. SEARCH METHODS: We searched the Cochrane Tobacco Addiction Group's Specialised Register, CENTRAL, MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature, and the trials registries clinicaltrials.gov and the International Clinical Trials Registry Platform. We ran all searches from database inception to 15 April 2021. SELECTION CRITERIA: We included cohort studies, and both cluster- and individually randomised controlled trials of at least six months' duration. We treated all included studies as cohort studies and analysed them by smoking status at follow-up. Eligible studies had to recruit adults (> 18 years) with diagnosed CHD and who smoked tobacco at diagnosis, and assess whether they quit or continued smoking during the study. Studies had to measure at least one of our included outcomes with at least six months' follow-up. Our primary outcomes were death from CVD and MACE. Secondary outcomes included all-cause mortality, non-fatal myocardial infarction, non-fatal stroke, new-onset angina and change in quality of life. DATA COLLECTION AND ANALYSIS: We followed standard Cochrane methods for screening and data extraction. We assessed the risk of bias for the primary outcomes using the ROBINS-I tool. We compared the incidence of death from CVD and of MACE (primary outcomes) between participants who quit smoking versus those who continued to smoke for each included study that reported these outcomes. We also assessed differences in all-cause mortality, incidence of non-fatal myocardial infarction, incidence of non-fatal stroke and new onset angina. We calculated hazard ratios (HRs) and 95% confidence intervals (95% CI). For our outcome, change in quality of life, we calculated the pooled standardised mean difference (SMD) and 95% CI for the difference in change in quality of life from baseline to follow-up between those who had quit smoking and those who had continued to smoke. For all meta-analyses we used a generic inverse variance random-effects model and quantified statistical heterogeneity using the I²statistic. We assessed the certainty of evidence for our primary outcomes using the eight GRADE considerations relevant to non-randomised studies. MAIN RESULTS: We included 68 studies, consisting of 80,702 participants. For both primary outcomes, smoking cessation was associated with a decreased risk compared with continuous smoking: CVD death (HR 0.61, 95% CI 0.49 to 0.75; I² = 62%; 18 studies, 17,982 participants; moderate-certainty evidence) and MACE (HR 0.57, 95% CI 0.45 to 0.71; I² = 84%; 15 studies, 20,290 participants; low-certainty evidence). These findings were robust to our planned sensitivity analyses. Through subgroup analysis, for example comparing adjusted versus non-adjusted estimates, we found no evidence of differences in the effect size. While there was substantial heterogeneity, this was primarily in magnitude rather than the direction of the effect estimates. Overall, we judged 11 (16%) studies to be at moderate risk of bias and 18 (26%) at serious risk, primarily due to possible confounding. There was also some evidence of funnel plot asymmetry for MACE outcomes. For these reasons, we rated our certainty in the estimates for CVD death as moderate and MACE as low. For our secondary outcomes, smoking cessation was associated with a decreased risk in all-cause mortality (HR 0.60, 95% CI 0.55 to 0.66; I² = 58%; 48 studies, 59,354 participants), non-fatal myocardial infarction (HR 0.64, 95% CI 0.58 to 0.72; I² = 2%; 24 studies, 23,264 participants) and non-fatal stroke (HR 0.70, 95% CI 0.53 to 0.90; I² = 0%; 9 studies, 11,352 participants). As only one study reported new onset of angina, we did not conduct meta-analysis, but this study reported a lower risk in people who stopped smoking. Quitting smoking was not associated with a worsening of quality of life and suggested improvement in quality of life, with the lower bound of the CI also consistent with no difference (SMD 0.12, 95% CI 0.01 to 0.24; I² = 48%; 8 studies, 3182 participants). AUTHORS' CONCLUSIONS: There is moderate-certainty evidence that smoking cessation is associated with a reduction of approximately one-third in the risk of recurrent cardiovascular disease in people who stop smoking at diagnosis. This association may be causal, based on the link between smoking cessation and restoration of endothelial and platelet function, where dysfunction of both can result in increased likelihood of CVD events. Our results provide evidence that there is a decreased risk of secondary CVD events in those who quit smoking compared with those who continue, and that there is a suggested improvement in quality of life as a result of quitting smoking. Additional studies that account for confounding, such as use of secondary CVD prevention medication, would strengthen the evidence in this area.
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Doenças Cardiovasculares , Doença das Coronárias , Infarto do Miocárdio , Abandono do Hábito de Fumar , Acidente Vascular Cerebral , Adulto , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/prevenção & controle , Doença das Coronárias/epidemiologia , Doença das Coronárias/prevenção & controle , Humanos , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/prevenção & controle , Qualidade de Vida , Prevenção Secundária , Abandono do Hábito de Fumar/métodos , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/prevenção & controleRESUMO
PURPOSE: Outcomes of patients with non-functioning pituitary adenomas categorized using the 2004 and 2017 WHO classification systems are understudied. We report outcomes from the University of Virginia of patients with non-functioning pituitary adenomas categorized using both systems. METHODS: We constructed a database from all 239 patients who underwent resection of a non-functioning pituitary adenoma between 2003 and 2015 and had at least 5 years of follow-up. Pathologic diagnosis was determined under both the 2004 and 2017 WHO classification systems. We compared the rates of recurrence and progression between subtypes using univariate and multivariate Cox regression analyses. RESULTS: Nearly 30% of the tumors in our database were classified as null cell adenomas under the 2004 classification system, whereas only 10% of the tumors were classified as null cell adenomas using the 2017 classification system. Most of these tumors were reclassified as either corticotroph or gonadotroph adenomas. Despite our relatively large cohort and average follow-up of nearly 9 years, we did not detect a significant difference in recurrence and progression between subtypes. CONCLUSIONS: The majority of null cell adenomas diagnosed under the 2004 WHO classification system are reclassified as gonadotroph or corticotroph adenomas under the 2017 WHO classification system. Rates of progression and recurrence between subtypes are not as different as previously believed at our institution and require a larger cohort to further investigate.
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Adenoma Hipofisário Secretor de ACT , Adenoma , Neoplasias Hipofisárias , Humanos , Neoplasias Hipofisárias/cirurgia , Neoplasias Hipofisárias/patologia , Adenoma/cirurgia , Adenoma/patologia , Adenoma Hipofisário Secretor de ACT/patologia , Organização Mundial da SaúdeRESUMO
BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has led to remarkable progress in our understanding of tissue heterogeneity in health and disease. Recently, the need for scRNA-seq sample fixation has emerged in many scenarios, such as when samples need long-term transportation, or when experiments need to be temporally synchronized. Methanol fixation is a simple and gentle method that has been routinely applied in scRNA-sEq. Yet, concerns remain that fixation may result in biases which may change the RNA-seq outcome. RESULTS: We adapted an existing methanol fixation protocol and performed scRNA-seq on both live and methanol fixed cells. Analyses of the results show methanol fixation can faithfully preserve biological related signals, while the discrepancy caused by fixation is subtle and relevant to library construction methods. By grouping transcripts based on their lengths and GC content, we find that transcripts with different features are affected by fixation to different degrees in full-length sequencing data, while the effect is alleviated in Drop-seq result. CONCLUSIONS: Our deep analysis reveals the effects of methanol fixation on sample RNA integrity and elucidates the potential consequences of using fixation in various scRNA-seq experiment designs.
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Metanol , RNA , Sequência de Bases , RNA/genética , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
BACKGROUND: Vascular patients with tissue loss requiring minor amputations could be an early sign of a terminal event. The long-term outcomes and timing of revascularisation for these patients are not well-studied. The aim of this study was to determine the clinical outcomes following minor amputations. Primary outcomes were functional status, limb loss, and mortality. Secondary outcomes compared immediate and delayed revascularisation. METHODS: A retrospective analysis of 200 vascular patients who required minor amputations at Austin Hospital, Melbourne was performed over 5 years. Demographics, details of revascularisation, functional status, and clinical outcomes such as recurrent tissue loss, limb loss and death were recorded. RESULTS: Of the entire cohort requiring minor amputations, 118 (59%) patients underwent revascularisation. 111 (94%) revascularisation procedures were performed within 90 days of minor amputation. Over all 5-year limb preservation was 89.9%. Patients who required revascularisation were not statistically significantly more at risk for limb loss at 5 years [13.6% vs. 6.6%; P=0.08]. Limb salvage at 1 year was not different between groups revascularized before and after amputation [89.5% vs. 90.9%; P=0.70]. Over all 5-year mortality rate was 50%. In the diabetic subset, those who had revascularisation after minor amputation had a greater 5-year mortality [67.9% vs. 50%; P=0.03]. A scoring system based on risk factors was developed but was not reliable based on the study data. CONCLUSIONS: The data from this study suggest that patients with diabetes who undergo revascularisation after minor amputation have worse outcomes than those revascularised prior to minor amputation. A predictive model applied at presentation could help detect high-risk patients but requires further work.
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Amputação Cirúrgica , Angiopatias Diabéticas/cirurgia , Doença Arterial Periférica/cirurgia , Procedimentos Cirúrgicos Vasculares , Idoso , Idoso de 80 Anos ou mais , Amputação Cirúrgica/efeitos adversos , Amputação Cirúrgica/mortalidade , Angiopatias Diabéticas/diagnóstico , Angiopatias Diabéticas/mortalidade , Feminino , Estado Funcional , Humanos , Salvamento de Membro , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/mortalidade , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Procedimentos Cirúrgicos Vasculares/mortalidade , VitóriaRESUMO
OBJECTIVES: To investigate whether anti-glypican-1 antibody Miltuximab conjugated with near-infrared dye IRDye800CW can be used for in vivo fluorescence imaging of urothelial carcinoma. METHODS: The conjugate, Miltuximab-IRDye800CW, was produced and characterized by size exclusion chromatography and flow cytometry with glypican-1-expressing cells. Balb/c nude mice bearing subcutaneous urothelial carcinoma xenografts were intravenously injected with Miltuximab-IRDye800CW or control IgG-IRDye800CW and imaged daily by fluorescence imaging. After 10 days, tumors and major organs were collected for ex vivo study of the conjugate biodistribution, including its accumulation in the tumor. RESULTS: The intravenous injection of Miltuximab-IRDye800CW to tumor-bearing mice showed its specific accumulation in the tumors with the tumor-to-background ratio of 12.7 ± 2.4, which was significantly higher than that in the control group (4.6 ± 0.9, P < 0.005). The ex vivo imaging was consistent with the in vivo findings, with tumors from the mice injected with Miltuximab-IRDye800CW being significantly brighter than the organs or the control tumors. CONCLUSIONS: The highly specific accumulation and retention of Miltuximab-IRDye800CW in glypican-1-expressing tumors in vivo shows its high potential for fluorescence imaging of urothelial carcinoma and warrants its further investigation toward clinical translation.
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Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Animais , Linhagem Celular Tumoral , Glipicanas , Camundongos , Camundongos Nus , Imagem Molecular , Imagem Óptica , Distribuição Tecidual , Neoplasias da Bexiga Urinária/diagnóstico por imagemRESUMO
Vascularization of engineered tissue constructs remains one of the greatest unmet challenges to mimicking the native tissue microenvironment in vitro. The main obstacle is recapitulating the complexity of the physiological environment while providing simplicity in operation and manipulation of the model. Microfluidic technology has emerged as a promising tool that enables perfusion of the tissue constructs through engineered vasculatures and precise control of the vascular microenvironment cues in vitro. The tunable microenvironment includes i) biochemical cues such as coculture, supporting matrix, and growth factors and ii) engineering aspects such as vasculature engineering methods, fluid flow, and shear stress. In this systematic review, the design considerations of the microfluidic-based in vitro model are discussed, with an emphasis on microenvironment control to enhance the development of next-generation vascularized engineered tissues.
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Microfluídica , Engenharia Tecidual , Técnicas de Cocultura , Humanos , Microfluídica/métodos , Neovascularização Patológica/patologia , Engenharia Tecidual/métodosRESUMO
AIMS: Renal cell carcinomas are relatively rare in children and young adults. While well characterised in adults, the morphological and molecular characterisation of these tumours in young patients is relatively lacking. The objective of this study was to explore the spectrum of renal cell carcinoma (RCC) subtypes in children and young adults and to determine their clinico-pathological, immunohistochemical and molecular characteristics by evaluating a large retrospective cohort of renal cell carcinoma patients age 30 years or younger. METHODS AND RESULTS: Sixty-eight cases with confirmed diagnosis of renal cell carcinoma at age 30 years or younger were identified at our institution. Clear cell carcinoma accounted for the most common subtype seen in this age group. Translocation renal cell carcinoma and rare familial syndrome subtypes such as succinate dehydrogenase deficient renal cell carcinoma and tuberous sclerosis complex-associated renal cell carcinoma were found relatively more frequently in this cohort. Despite applying the 2016 WHO classification criteria, a high proportion of the tumours in our series remained unclassified. CONCLUSIONS: Our results suggest that renal cell carcinoma in children and young adults is a relatively rare disease that shares many histological similarities to renal cell carcinoma occurring in adults and yet demonstrate some unique clinical-pathological differences. Microphthalmia-associated transcription (MiT) family translocation RCC and rare familial syndrome subtypes are relatively more frequent in the paediatric and adolescent age groups than in adults. Clear cell RCC still accounted for the most common subtype seen in this age group. MiT family translocation RCC patients presented with advanced stage disease and had poor clinical outcomes. The large and heterogeneous subgroup of unclassified renal cell carcinoma contains phenotypically distinct tumours with further potential for future subcategories in the renal cell carcinoma classification.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Adolescente , Adulto , Idade de Início , Criança , Feminino , Humanos , Masculino , Adulto JovemRESUMO
The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.