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Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives.
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Enfermedades Transmisibles , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reproducibilidad de los Resultados , Metagenoma , Estándares de Referencia , Metagenómica , Enfermedades Transmisibles/diagnósticoRESUMEN
Circulating tumor RNA (ctRNA) has recently emerged as a novel and attractive liquid biomarker. CtRNA is capable of providing important information about the expression of a variety of target genes noninvasively, without the need for biopsies, through the use of circulating RNA sequencing. The overexpression of cancer-specific transcripts increases the tumor-derived RNA signal, which overcomes limitations due to low quantities of circulating tumor DNA (ctDNA). The purpose of this work is to present an up-to-date review of current knowledge regarding ctRNAs and their status as biomarkers to address the diagnosis, prognosis, prediction, and drug resistance of colorectal cancer. The final section of the article discusses the practical aspects involved in analyzing plasma ctRNA, including storage and isolation, detection technologies, and their limitations in clinical applications.
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Ácidos Nucleicos Libres de Células , ADN Tumoral Circulante , Neoplasias Colorrectales , Humanos , Biopsia Líquida , Ácidos Nucleicos Libres de Células/genética , Biomarcadores de Tumor/genética , ARN/genética , Neoplasias Colorrectales/patologíaRESUMEN
Background: Cell free RNA (cfRNA) contains transcript fragments from multiple cell types, making it useful for cancer detection in clinical settings. However, the pathophysiological origins of cfRNAs in plasma from colorectal cancer (CRC) patients remain unclear. Methods: To identify the tissue-specific contributions of cfRNAs transcriptomic profile, we used a published single-cell transcriptomics profile to deconvolute cell type abundance among paired plasma samples from CRC patients who underwent tumor-ablative surgery. We further validated the differentially expressed cfRNAs in 5 pairs of CRC tumor samples and adjacent tissue samples as well as 3 additional CRC tumor samples using RNA-sequencing. Results: The transcriptomic component from intestinal secretory cells was significantly decreased in the in-house post-surgical cfRNA. The HPGD, PACS1, and TDP2 expression was consistent across cfRNA and tissue samples. Using the Cancer Genome Atlas (TCGA) CRC datasets, we were able to classify the patients into two groups with significantly different survival outcomes. Conclusions: The three-gene signature holds promise in applying minimal residual disease (MRD) testing, which involves profiling remnants of cancer cells after or during treatment. Biomarkers identified in the present study need to be validated in a larger cohort of samples in order to ascertain their possible use in early diagnosis of CRC.
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Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.
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COVID-19 , Adulto , Humanos , Niño , Filogenia , SARS-CoV-2 , Genoma ViralRESUMEN
The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases.
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COVID-19 , Humanos , COVID-19/diagnóstico , Pruebas Genéticas/métodos , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Consenso , Prueba de COVID-19RESUMEN
BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths in Hong Kong. We tested the hypothesis that circulating tumor cell (CTC) analysis by ARB101 antibody could be used as a tool for CRC detection, progression, and therapy response. RESEARCH METHODS: ARB101 antibody was used for investigation of CDH17 expression in formalin-fixed, paraffin-embedded (FFPE) tissue sections and circulating tumor cells (CTCs) of CRC patients. RESULTS: Using ARB101, highest sensitivity was observed in 98/100 (98%) colorectal cancer tissue compared to 72/100 gastric cancer (72%) and 27/32 pancreatic cancer (84%). Immunoreactivity of CDH17 was significantly higher in distant metastatic (tumor-node-metastasis [TNM] stage IV) than non-distant metastatic (TNM stage I to III) CRC. ARB101 antibody also manifested the higher sensitivity than c-erbB2 (8%) and epidermal growth factor receptor (EGFR)-targeting antibodies (37%) with the significance (p < 0.0001). ARB101 positive CTCs were detected in 64/83 (77%) TNM stage I to IV CRC patients. Furthermore, ARB101 positive CTCs detected in TNM stage I to III CRC patients before and after surgical operation are statistically significant (p < 0.0001). CONCLUSIONS: CTC detection by ARB101 antibody could serve as a potential non-invasive approach for CRC detection, progression, and monitoring of treatment response.
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Neoplasias Colorrectales , Células Neoplásicas Circulantes , Neoplasias Pancreáticas , Neoplasias Gástricas , Humanos , Células Neoplásicas Circulantes/patología , Neoplasias Colorrectales/metabolismo , Hong Kong , Biomarcadores de Tumor/metabolismo , CadherinasRESUMEN
Colorectal cancer (CRC) threatens human health seriously. Early diagnosis of CRC is critical to improving patient survival. Meanwhile, non-invasive detection through tumor-circulating markers can be an important auxiliary diagnosis. In this study, we performed targeted RNA sequencing in paired tumor and adjacent normal fresh frozen tissues from 68 patients, and we also measured circulating mRNA levels in 4 time-point plasma samples collected before and after operation or chemotherapy. Our results showed that SOX9 (6.73-fold with adjusted p value < 1 × 10-45), MYC (20.59-fold with adjusted p value < 1 × 10-57), and MMP7 (131.94-fold with adjusted p value < 1 × 10-78) highly expressed in tumor compared with adjacent normal tissues. Besides, the circulating mRNA of SOX9 (41.14-fold with adjusted p value < 1 × 10-13) in CRC was significantly higher than in the normal control as well. Moreover, a SOX9-based 9-gene panel (SOX9, GSK3A, FZD4, LEF1, DVL1, FZD7, NFATC1, KRT19, and RUVBL1) showed the non-invasive diagnostic value of CRC (AUC: 0.863 (0.766-0.960), TPR: 0.92, TNR: 0.87). In summary, SOX9 expression consistently increases in tumor and plasma samples from CRC patients, which indicates the important role of SOX9 in CRC progression and its potential in non-invasive diagnosis of CRC.
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Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Biomarcadores de Tumor , Detección Precoz del Cáncer/métodos , ARN Mensajero , Regulación Neoplásica de la Expresión Génica , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
INTRODUCTION: Clinical metagenomic next-generation sequencing (mNGS) allows a comprehensive genetic analysis of microbial materials. Different from other traditional target-driven molecular diagnostic tests, such as PCR, mNGS is a hypothesis-free diagnostic approach that allows a comprehensive genetic analysis of the clinical specimens that cover nearly any common, rare, and new pathogens ranging broadly from viruses, bacteria, fungi to parasites. AREAS COVERED: In this article, we discussed the clinical application of the mNGS using two clinical cases as examples and described the use of mNGS to assist the diagnosis of parasitic pulmonary infection. The advantages and challenges in implementing mNGS in clinical microbiology are also discussed. EXPERT OPINION: mNGS is a promising technology that allows quick diagnosis of infectious diseases. Currently, a plethora of sequencing and analysis methods exists for mNGS, each with individual merits and pitfalls. While standards and best practices were proposed by various metagenomics working groups, they are yet to be widely adopted in the community. The development of a consensus set of guidelines is necessary to guide the usage of this new technology and the interpretation of NGS results before clinical adoption of mNGS testing.
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Enfermedades Transmisibles , Metagenómica , Líquido del Lavado Bronquioalveolar/microbiología , Enfermedades Transmisibles/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Metagenoma , Metagenómica/métodos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The import of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.36.27 has sparked the fourth wave of COVID-19 outbreak in Hong Kong. This strain has been circulating in Hong Kong since September 2020 but rarely found in other countries (<1%). RESEARCH DESIGN AND METHODS: A total of 14 SARS-CoV-2 genome sequences collected from patients in Hong Kong between July 2020 and March 2021 were determined by whole viral genome sequencing using Illumina next-generation sequencing platform, followed by phylogenetic analysis. RESULTS: Of the 14 SARS-CoV-2 genome sequences analyzed, 9 strains belonged to the PANGO lineage B.1.36.27, GISAID clade GH, and Nextclade clade 20A. Compared to the reference genome, 31 nucleotide differences and 11 amino acid differences were identified in the genome of the SARS-CoV-2 from PANGO lineage B.1.36.27. CONCLUSIONS: We reported the nucleotides and amino acids mutations identified in the SARS-CoV-2 from PANGO lineage B.1.36.27. Our viral genome sequences enriched the understanding of SARS-CoV-2 mutational landscape and improved the repertoire of known SARS-CoV-2 variants for tracking and tracing. From this study, we found no evidence to show that SARS-CoV-2 from lineage B.1.36.27 can compromise existing vaccines and antibody therapies.
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Genoma Viral , Filogenia , SARS-CoV-2 , COVID-19/virología , Hong Kong/epidemiología , Humanos , SARS-CoV-2/genéticaRESUMEN
INTRODUCTION: To date, the transmission of Coronavirus Disease-2019 (COVID-19) is still uncontrollable with the fact that the numbers of confirmed and death cases are still increasing. Up to 1st October 2020, 33,842,281 confirmed cases and 1,010,634 confirmed deaths have been reported to the World Health Organization from 216 different countries, areas and territories. Despite the urgent demand for effective treatment strategies, there is still no specific antiviral treatment for COVID-19 and the treatment guidelines for COVID-19 vary between countries. AREA COVERED: In this article, we summarized the current knowledge on COVID-19 and the pandemic worldwide. Moreover, the epidemiology, pathogenesis, prevention and different treatment options will be discussed so that we shall prepare ourselves better to fight with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). EXPERT OPINION: The situation of the COVID-19 pandemic is still unpredictable. There is no effective vaccine or specific anti-viral drug to treat serve COVID-19 patients. Combination therapies have shown promising clinical improvement. Repurposing FDA-approved drugs might be one of possible treatment options. Without specific treatment and vaccines for COVID-19, the most effective way to prevent from being infected is to generate an ecosystem with effective protection, precautions and preventive measures.
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Tratamiento Farmacológico de COVID-19 , COVID-19/epidemiología , Animales , Antivirales/administración & dosificación , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Reposicionamiento de Medicamentos , HumanosRESUMEN
INTRODUCTION: There are great potentials of using exosomal RNAs (exoRNA) as biomarkers in cancers. The isolation of exoRNA requires the use of ultracentrifugation to isolate cell-free RNA followed by detection using real-time PCR, microarray, next-generation sequencing, or Nanostring nCounter system. The use of exoRNA enrichment panels has largely increased the detection sensitivity and specificity when compared to traditional diagnostic tests. Moreover, using exoRNA as biomarkers can assist the early detection of chemo and radioresistance cancer, and in turn opens up the possibility of personalized treatment to patients. Finally, exoRNA can be detected at an early stage of cancer recurrence to improve the survival rate. AREAS COVERED: In this review, the authors summarized the detection methods of exoRNA as well as its potential as a biomarker in cancer diagnosis and chemo and radioresistance. EXPERT OPINION: The application of exoRNAs in clinical diagnosis is still in its infancy. Further researches on extracellular vesicles isolation, detection protocols, exoRNA classes and subclasses, and the regulatory biological pathways have to be performed before exoRNA can be applied translationally.
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Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/sangre , Exosomas/química , Neoplasias/sangre , ARN Neoplásico/sangre , Secuencia de Bases , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma/sangre , Carcinoma/diagnóstico , Carcinoma/patología , Carcinoma/terapia , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Resistencia a Antineoplásicos , Detección Precoz del Cáncer/métodos , Femenino , Citometría de Flujo/métodos , Humanos , Masculino , MicroARNs/sangre , MicroARNs/aislamiento & purificación , Análisis por Micromatrices , Nanotecnología/instrumentación , Nanotecnología/métodos , Estadificación de Neoplasias/métodos , Neoplasias/genética , Pronóstico , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Ultracentrifugación/métodosRESUMEN
To obtain a comprehensive understanding of transcriptomic reprogramming under salt stress, we performed whole-transcriptome sequencing on the leaf and root of soybean seedlings subjected to salt treatment in a time-course experiment (0, 1, 2, 4, 24, and 48 hr). This time series dataset enabled us to identify important hubs and connections of gene expressions. We highlighted the analysis on phytohormone signaling pathways and their possible crosstalks. Differential expressions were also found among those genes involved in carbon and nitrogen metabolism. In general, the salt-treated seedlings slowed down their photosynthetic functions and ramped up sugar catabolism to provide extra energy for survival. Primary nitrogen assimilation was shut down whereas nitrogen resources were redistributed. Overall, the results from the transcriptomic analyses indicate that the plant uses a multipronged approach to overcome salt stress, with both fast-acting, immediate physiological responses, and longer term reactions that may involve metabolic adjustment.
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Regulación de la Expresión Génica de las Plantas , Glycine max/metabolismo , Estrés Salino , Plantones/metabolismo , Perfilación de la Expresión Génica , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , Estrés Salino/fisiología , Plantones/fisiología , Glycine max/fisiologíaRESUMEN
Genetic testing for neurodegenerative diseases (NDs) is highly challenging because of genetic heterogeneity and overlapping manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington's disease and spinocerebellar ataxias type 3 (SCA3). Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known or likely pathogenic variation was found in 54 patients; 27 patients demonstrated clinical profiles that matched the variants; and 16 patients whose original diagnosis were refined. A high concordance of variant calling were observed when comparing the results from TGP and whole-exome sequencing of four patients. Our in-house STR detection algorithm has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combined comprehensive TGPs-bioinformatics pipeline can improve the clinical diagnosis of NDs.
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Purpose To determine if sex differences in abdominal visceral fat composition, measured by using computed tomography (CT), and tumor glucose metabolism, measured by gene expression, can help predict outcomes in patients with clear cell renal cell carcinoma (RCC). Materials and Methods This retrospective cohort study included 222 patients with clear cell RCC from The Cancer Imaging Atlas. By using CT, body fat was segmented into subcutaneous fat and visceral fat areas (VFAs) and normalized to total fat to obtain the relative VFA (rVFA) and relative subcutaneous fat area. Multivariate Cox proportional hazard regression models were performed to identify effects of rVFA on sex-specific survival. Expression profiles for 39 glycolytic genes in tumors from these patients were obtained from The Cancer Genome Atlas to determine sex differences in metabolism and compared with rVFA. Key mutations in clear cell RCC were analyzed for association with rVFA and tumor glycolytic profiles. Results Women with rVFA greater than 30.9% had an increased risk of death (hazard ratio, 3.66 [95% confidence interval: 1.64, 8.19]) for women vs 1.13 ([95% confidence interval: 0.58, 2.18] for men, P = .028). Glycolytic gene expression stratified both men and women, and the combination of low rVFA and low glycolysis identified 19 women with excellent overall survival (P < .001). SETD2 and BAP1 mutations were uniquely enriched in female tumors with high glycolysis (P = .036 and .001, respectively). No significant differences were identified in tumor mutations between patients with high and low rVFA. Conclusion Sex differences in visceral fat and tumor glucose metabolism may provide a new risk-stratification system for patients with clear cell RCC. © RSNA, 2018 Online supplemental material is available for this article.
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Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/metabolismo , Grasa Intraabdominal/diagnóstico por imagen , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/metabolismo , Tomografía Computarizada por Rayos X/métodos , Estudios de Cohortes , Femenino , Glucosa/metabolismo , Humanos , Riñón/diagnóstico por imagen , Riñón/metabolismo , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores Sexuales , Análisis de SupervivenciaRESUMEN
Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance.
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Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica , Transcriptoma , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/aislamiento & purificación , Amicacina/farmacología , Elementos Transponibles de ADN , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Imipenem/farmacología , Meropenem/farmacología , Anotación de Secuencia MolecularRESUMEN
The Toll/interleukin-1 receptor (TIR) domain is the signature signaling domain of Toll-like receptors (TLRs) and their adaptors, serving as a scaffold for the assembly of protein complexes for innate immune signaling [1, 2]. TIR domain proteins are also expressed in plants, where they mediate disease resistance [3, 4], and in bacteria, where they have been associated with virulence [5-9]. In pursuing our work on axon degeneration [10], we made the surprising discovery that the TIR domain of SARM1 (sterile alpha and TIR motif containing 1), a TLR adaptor protein, has enzymatic activity [11]. Upon axon injury, the SARM1 TIR domain cleaves nicotinamide adenine dinucleotide (NAD+), destroying this essential metabolic co-factor to trigger axon destruction [11, 12]. Whereas current studies of TIR domains focus on their scaffolding function, our findings with SARM1 inspired us to ask whether this enzymatic activity is the primordial function of the TIR domain. Here we show that ancestral prokaryotic TIR domains constitute a new family of NADase enzymes. Using purified proteins from a cell-free translation system, we find that TIR domain proteins from both bacteria and archaea cleave NAD+ into nicotinamide and ADP-ribose (ADPR), with catalytic cleavage executed by a conserved glutamic acid. A subset of bacterial and archaeal TIR domains generates a non-canonical variant cyclic ADPR (cADPR) molecule, and the full-length TIR domain protein from pathogenic Staphylococcus aureus induces NAD+ loss in mammalian cells. These findings suggest that the primordial function of the TIR domain is the enzymatic cleavage of NAD+ and establish TIR domain proteins as a new class of metabolic regulatory enzymes.
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Archaea/genética , Proteínas Arqueales/genética , Bacterias/genética , Proteínas Bacterianas/genética , Animales , Archaea/enzimología , Proteínas Arqueales/metabolismo , Axones/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , RatonesRESUMEN
The molecular bases for sex differences in cancer remain undefined and how to incorporate them into risk stratification remains undetermined. Given sex differences in metabolism and the inverse correlation between fluorodeoxyglucose (FDG) uptake and survival, we hypothesized that glycolytic phenotyping would improve glioma subtyping. Using retrospectively acquired lower-grade glioma (LGG) transcriptome data from The Cancer Genome Atlas (TCGA), we discovered male-specific decreased survival resulting from glycolytic gene overexpression. Patients within this high-glycolytic group showed significant differences in the presence of key genomic alterations (i.e., 1p/19q codeletion, CIC, EGFR, NF1, PTEN, FUBP1, and IDH mutations) compared with the low-glycolytic group. Although glycolytic stratification defined poor prognostic males independent of grade, histology, TP53, and ATRX mutation status, we unexpectedly found that females with high-glycolytic gene expression and wild-type IDH survived longer than all other wild-type patients. Validation with an independent metabolomics dataset from grade 2 gliomas determined that glycolytic metabolites selectively stratified males and also uncovered a potential sexual dimorphism in pyruvate metabolism. These findings identify a potential synergy between patient sex, tumor metabolism, and genomic alterations in determining outcome for glioma patients.
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Small RNAs, including microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs; from PHAS loci), play key roles in plant development. Cultivated soybean, Glycine max, contributes a great deal to food production, but, compared to its wild kin, Glycine soja, it may lose some genetic information during domestication. In this work, we analyzed the sRNA profiles of different tissues in both cultivated (C08) and wild soybeans (W05) at three stages of development. A total of 443 known miRNAs and 15 novel miRNAs showed varying abundances between different samples, but the miRNA profiles were generally similar in both accessions. Based on a sliding window analysis workflow that we developed, 50 PHAS loci generating 55 21-nucleotide phasiRNAs were identified in C08, and 46 phasiRNAs from 41 PHAS loci were identified in W05. In germinated seedlings, phasiRNAs were more abundant in C08 than in W05. Disease resistant TIR-NB-LRR genes constitute a very large family of PHAS loci. PhasiRNAs were also generated from several loci that encode for NAC transcription factors, Dicer-like 2 (DCL2), Pentatricopeptide Repeat (PPR), and Auxin Signaling F-box 3 (AFB3) proteins. To investigate the possible involvement of miRNAs in initiating the PHAS-phasiRNA pathway, miRNA target predictions were performed and 17 C08 miRNAs and 15 W05 miRNAs were predicted to trigger phasiRNAs biogenesis. In summary, we provide a comprehensive description of the sRNA profiles of wild versus cultivated soybeans, and discuss the possible roles of sRNAs during soybean germination.
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Fabaceae/genética , Glycine max/genética , ARN de Planta/genética , Regulación de la Expresión Génica de las Plantas/genética , MicroARNs/genética , ARN Interferente PequeñoRESUMEN
Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq) datasets (26 sequencing libraries in total) to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples) on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.