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The outbreak of COVID-19 has created an unprecedent global crisis. While the polymerase chain reaction (PCR) is the gold standard method for detecting active SARS-CoV-2 infection, alternative high-throughput diagnostic tests are of a significant value to meet universal testing demands. Here, we describe a new design of the MasSpec Pen technology integrated to electrospray ionization (ESI) for direct analysis of clinical swabs and investigate its use for COVID-19 screening. The redesigned MasSpec Pen system incorporates a disposable sampling device refined for uniform and efficient analysis of swab tips via liquid extraction directly coupled to an ESI source. Using this system, we analyzed nasopharyngeal swabs from 244 individuals including symptomatic COVID-19 positive, symptomatic negative, and asymptomatic negative individuals, enabling rapid detection of rich lipid profiles. Two statistical classifiers were generated based on the lipid information acquired. Classifier 1 was built to distinguish symptomatic PCR-positive from asymptomatic PCR-negative individuals, yielding a cross-validation accuracy of 83.5%, sensitivity of 76.6%, and specificity of 86.6%, and validation set accuracy of 89.6%, sensitivity of 100%, and specificity of 85.3%. Classifier 2 was built to distinguish symptomatic PCR-positive patients from negative individuals including symptomatic PCR-negative patients with moderate to severe symptoms and asymptomatic individuals, yielding a cross-validation accuracy of 78.4%, specificity of 77.21%, and sensitivity of 81.8%. Collectively, this study suggests that the lipid profiles detected directly from nasopharyngeal swabs using MasSpec Pen-ESI mass spectrometry (MS) allow fast (under a minute) screening of the COVID-19 disease using minimal operating steps and no specialized reagents, thus representing a promising alternative high-throughput method for screening of COVID-19.
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COVID-19 , Testes Diagnósticos de Rotina , Humanos , Nasofaringe , SARS-CoV-2 , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.
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Ácido Aspártico/química , Caspases/química , Caspases/metabolismo , Nicotiana/enzimologia , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Sítios de Ligação , Caspases/genética , DNA Complementar , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência , Nicotiana/genética , Trypanosoma brucei brucei/enzimologiaRESUMO
INTRODUCTION: Studies addressing the methylation pattern in adamantinomatous craniopharyngioma (ACP) are lacking. OBJECTIVE: To identify methylation signatures in ACPs regarding clinical presentation and outcome. METHODS: Clinical and pathology data were collected from 35 ACP patients (54% male; 18.1 years [2-68]). CTNNB1 mutations and methylation profile (MethylationEPIC/Array-Illumina) were analyzed in tumoral DNA. Unsupervised machine learning analysis of this comprehensive methylome sample was achieved using hierarchical clustering and multi-dimensional scaling. Statistical associations between clusters and clinical features were achieved using Fisher's test and global biological process interpretations were aided by Gene Ontology enrichment analyses. RESULTS: Two clusters were revealed consistently by all unsupervised methods (ACP-1: n = 18; ACP-2: n = 17) with strong bootstrap statistical support. ACP-2 was enriched by CTNNB1 mutations (100% vs 56%, P = 0.0006), hypomethylated in CpG Island (CGI), non-CGI sites, and globally (P < 0.001), and associated with greater tumor size (24.1 vs 9.5cm3, P = 0.04). Enrichment analysis highlighted pathways on signaling transduction, transmembrane receptor, development of anatomical structures, cell-adhesion, cytoskeleton organization, and cytokine binding, and also cell-type specific biological processes as regulation of oligodendrocytes, keratinocyte, and epithelial cells differentiation. CONCLUSION: Two clusters of ACP patients were consistently revealed by unsupervised machine learning methods, being one of them significantly hypomethylated, enriched by CTNNB1 mutated ACPs, and associated with increased tumor size. Enrichment analysis reinforced pathways involved in tumor proliferation and in cell-specific tumoral microenvironment.
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OBJECTIVE: To explore pituitary tumors by methylome and transcriptome signatures in a heterogeneous ethnic population. METHODS: In this retrospective cross-sectional study, clinicopathological features, methylome, and transcriptome were evaluated in pituitary tumors from 77 patients (61% women, age 12-72 years) followed due to functioning (FPT: GH-secreting n = 18, ACTH-secreting n = 14) and nonfunctioning pituitary tumors (NFPT, n = 45) at Ribeirao Preto Medical School, University of São Paulo. RESULTS: Unsupervised hierarchical clustering analysis (UHCA) of methylome (n = 77) and transcriptome (n = 65 out of 77) revealed 3 clusters each: one enriched by FPT, one by NFPT, and a third by ACTH-secreting and NFPT. Comparison between each omics-derived clusters identified 3568 and 5994 differentially methylated and expressed genes, respectively, which were associated with each other, with tumor clinical presentation, and with 2017 and 2022 WHO classifications. UHCA considering 11 transcripts related to pituitary development/differentiation also supported 3 clusters: POU1F1-driven somatotroph, TBX19-driven corticotroph, and NR5A1-driven gonadotroph adenomas, with rare exceptions (NR5A1 expressed in few GH-secreting and corticotroph silent adenomas; POU1F1 in few ACTH-secreting adenomas; and TBX19 in few NFPTs). CONCLUSION: This large heterogenic ethnic Brazilian cohort confirms that integrated methylome and transcriptome signatures classify FPT and NFPT, which are associated with clinical presentation and tumor invasiveness. Moreover, the cluster NFPT/ACTH-secreting adenomas raises interest regarding tumor heterogeneity, supporting the challenge raised by the 2017 and 2022 WHO definition regarding the discrepancy, in rare cases, between clinical presentation and pituitary lineage markers. Finally, making our data publicly available enables further studies to validate genes/pathways involved in pituitary tumor pathogenesis and prognosis.
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Adenoma Hipofisário Secretor de ACT , Adenoma , Neoplasias Hipofisárias , Humanos , Feminino , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Masculino , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Adenoma/genética , Adenoma/patologia , Epigenoma , Transcriptoma , Estudos Retrospectivos , Estudos Transversais , Adenoma Hipofisário Secretor de ACT/genética , Hormônio Adrenocorticotrópico/genéticaRESUMO
OBJECTIVE: To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carcinoma (ACC) cells. METHODS: We evaluated VDR's methylation pattern in H295R ACC cells, and investigated the effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis. RESULTS: VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects. CONCLUSIONS: H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient's clinical management.
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Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/genética , Calcitriol/farmacologia , Carcinogênese/genética , Cateninas/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Hormônios/farmacologia , Receptores de Calcitriol/genética , Vitamina D/farmacologia , Via de Sinalização WntRESUMO
Children diagnosed with pediatric adrenocortical tumors (pACT) have variable outcomes, and, to date, the disease lacks robust prognostic biomarkers. The prognostic potential of tumor methylation has been demonstrated in several cancers. We aimed to evaluate the pACT methylation profile and its association with disease presentation and survival. In this cross-sectional study, we accessed the DNA methylation (MethylationEPIC Array, Illumina) of 57 primary pACT from Southeastern Brazil and the respective patients' clinicopathological features. We also applied our analysis in an independent 48 pACT methylation dataset. Unsupervised learning whole-methylome analysis showed two groups with distinct methylation signatures: pACT-1 and pACT-2. Compared to pACT-2, pACT-1 tumors were enriched with higher methylation in CpG islands, mainly in gene promoter regions. The topmost hypermethylated gene in these samples was shown to be underexpressed. Patients in the pACT-1 group were older at diagnosis and were more likely to have carcinomas and nonlocalized/advanced and recurrent/metastatic disease. Univariate and bivariate regressions showed that pACT-1 methylation signature confers superior hazard ratio of disease progression and death than known prognostic features. The methylation groups had similar frequencies of germline mutations in the TP53 gene, including the regionally frequent p.R337H. Our analysis replication validated our findings and reproduced those recently described in pACT. We demonstrated the existence of different tumor methylation signatures associated with pACT presentation and clinical evolution, even in the context of germline TP53 mutations. Our data support tumor methylation profiling as a robust and independent prognostic biomarker for pACT and suggest a list of candidate genes for further validation.
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Neoplasias do Córtex Suprarrenal , Metilação de DNA , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Biomarcadores , Biomarcadores Tumorais/genética , Criança , Ilhas de CpG , Estudos Transversais , Humanos , PrognósticoRESUMO
Objective: Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design: Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods: We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results: Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions: VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.