RESUMO
RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.
Assuntos
Capuzes de RNA/genética , Infecções por Vírus de RNA/genética , Proteínas Recombinantes de Fusão/genética , Regiões 5' não Traduzidas/genética , Animais , Bovinos , Linhagem Celular , Cricetinae , Cães , Humanos , Vírus da Influenza A/metabolismo , Camundongos , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Fases de Leitura Aberta/genética , Capuzes de RNA/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
It is now established that many viruses that threaten public health establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol and reduces genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.
Assuntos
Vírus da Influenza A , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Vírus da Influenza A/genética , Microtúbulos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND: Tobacco use is one of the main risk factors for Lung Cancer (LC) development. However, about 10-20% of those diagnosed with the disease are never-smokers. For Non-Small Cell Lung Cancer (NSCLC) there are clear differences in both the clinical presentation and the tumor genomic profiles between smokers and never-smokers. For example, the Lung Adenocarcinoma (LUAD) histological subtype in never-smokers is predominately found in young women of European, North American, and Asian descent. While the clinical presentation and tumor genomic profiles of smokers have been widely examined, never-smokers are usually underrepresented, especially those of a Latin American (LA) background. In this work, we characterize, for the first time, the difference in the genomic profiles between smokers and never-smokers LC patients from Chile. METHODS: We conduct a comparison by smoking status in the frequencies of genomic alterations (GAs) including somatic mutations and structural variants (fusions) in a total of 10 clinically relevant genes, including the eight most common actionable genes for LC (EGFR, KRAS, ALK, MET, BRAF, RET, ERBB2, and ROS1) and two established driver genes for malignancies other than LC (PIK3CA and MAP2K1). Study participants were grouped as either smokers (current and former, n = 473) or never-smokers (n = 200) according to self-report tobacco use at enrollment. RESULTS: Our findings indicate a higher overall GA frequency for never-smokers compared to smokers (58 vs. 45.7, p-value < 0.01) with the genes EGFR, KRAS, and PIK3CA displaying the highest prevalence while ERBB2, RET, and ROS1 the lowest. Never-smokers present higher frequencies in seven out of the 10 genes; however, smokers harbor a more complex genomic profile. The clearest differences between groups are seen for EGFR (15.6 vs. 21.5, p-value: < 0.01), PIK3CA (6.8 vs 9.5) and ALK (3.2 vs 7.5) in favor of never-smokers, and KRAS (16.3 vs. 11.5) and MAP2K1 (6.6 vs. 3.5) in favor of smokers. Alterations in these genes are comprised almost exclusively by somatic mutations in EGFR and mainly by fusions in ALK, and only by mutations in PIK3CA, KRAS and MAP2K1. CONCLUSIONS: We found clear differences in the genomic landscape by smoking status in LUAD patients from Chile, with potential implications for clinical management in these limited-resource settings.
Assuntos
Neoplasias Pulmonares , não Fumantes , Fumantes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/etiologia , Feminino , Masculino , Fumantes/estatística & dados numéricos , Pessoa de Meia-Idade , não Fumantes/estatística & dados numéricos , Idoso , Fumar/genética , Fumar/efeitos adversos , Fumar/epidemiologia , Mutação , Genômica/métodos , Adulto , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/patologiaRESUMO
Asthma drug responses may differ due to inflammatory mechanisms triggered by the immune cells in the pulmonary microenvironment. Thus, asthma phenotyping based on the local inflammatory profile may aid in treatment definition and the identification of new therapeutic targets. Here, we investigated protein profiles of induced sputum and serum from asthma patients classified into eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic asthma, according to inflammatory phenotypes. Proteomic analyses were performed using an ultra-performance liquid chromatography (ultra-HPLC) system coupled to the Q Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. Fifty-two (52) proteins showed significant differences in induced sputum among the groups, while only 12 were altered in patients' sera. Five proteins in the induced sputum were able to discriminate all phenotypic groups, while four proteins in the serum could differentiate all except the neutrophilic from the paucigranulocytic inflammatory pattern. This is the first report on comparative proteomics of inflammatory asthma phenotypes in both sputum and serum samples. We have identified a potential five-biomarker panel that may be able to discriminate all four inflammatory phenotypes in sputum. These findings not only provide insights into potential therapeutic targets but also emphasize the potential for personalized treatment approaches in asthma management.
Assuntos
Asma , Escarro , Humanos , Neutrófilos/metabolismo , Proteômica , Inflamação/metabolismo , Fenótipo , EosinófilosRESUMO
Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαßDAI), which harbours two Zα domains, and for a ZαßDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαßDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαßDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.
Assuntos
DNA Forma Z , Ácidos Nucleicos , Adenosina Desaminase/metabolismo , Grânulos Citoplasmáticos/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA , Proteínas de Ligação a RNARESUMO
Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.
Assuntos
Antígenos CD55/fisiologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Pulmão/imunologia , Viremia/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos CD55/química , Antígenos CD55/deficiência , Quimiotaxia de Leucócito , Ativação do Complemento , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Adaptação ao Hospedeiro , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Interferon gama/análise , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico , Neuraminidase/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Carga Viral , Proteínas Virais/fisiologia , Virulência , Replicação Viral , Redução de PesoRESUMO
Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.
Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos/genética , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Linhagem Celular , Humanos , Evasão da Resposta Imune , Mutação/genética , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
ObjectiveThis observational case-control study analyzed the clinical, functional, inflammatory profile, and treatment data of a cohort of patients with asthma who were followed up at the outpatient clinic of a teaching hospital.MethodsPatients who visited the clinic between January 2008 and February 2020 and diagnosed with asthma according to the Global Initiative for Asthma (GINA) criteria were included in the study. Patients were broadly classified into two groups: age <60 or age ≥60 years. The patients were evaluated for asthma control and severity, medications used, comorbidities, smoking status, occurrence of exacerbation, spirometry at the first and last visits, sputum cytology, allergic prick test, and inflammatory cytokine levels.ResultsPatients over 60 years of age had lower asthma control test (ACT) scores, required higher doses of inhaled corticosteroids to achieve asthma control and had worse lung function with fixed airway obstruction, higher number of comorbidities, greater exposure to tobacco, and longer outpatient follow-up than younger patients with asthma. Furthermore, older patients presented with neutrophilia and higher levels of TNFα in the induced sputum as compared to younger patients.ConclusionsThese findings suggest that patients aged ≥60 years of age had a more severe asthma profile and poorer lung function than younger patients with asthma. Furthermore, aging, long-term asthma, comorbidities, and tobacco exposure contributed to an accelerated decline in lung function.
Assuntos
Obstrução das Vias Respiratórias , Asma , Adulto , Humanos , Pessoa de Meia-Idade , Idoso , Asma/tratamento farmacológico , Estudos de Casos e Controles , Pulmão , ComorbidadeRESUMO
Biomolecular condensates are crucial compartments within cells, relying on their material properties for function. They form and persist through weak, transient interactions, often undetectable by classical biochemical approaches. Hence, microscopy-based techniques have been the most reliable methods to detail the molecular mechanisms controlling their formation, material properties, and alterations, including dissolution or phase transitions due to cellular manipulation and disease, and to search for novel therapeutic strategies targeting biomolecular condensates. However, technical challenges in microscopy-based analysis persist. This paper discusses imaging, data acquisition, and analytical methodologies' advantages, challenges, and limitations in determining biophysical parameters explaining biomolecular condensate formation, dissolution, and phase transitions. In addition, we mention how machine learning is increasingly important for efficient image analysis, teaching programs what a condensate should resemble, aiding in the correlation and interpretation of information from diverse data sources. Influenza A virus forms liquid viral inclusions in the infected cell cytosol that serve as model biomolecular condensates for this study. Our previous work showcased the possibility of hardening these liquid inclusions, potentially leading to novel antiviral strategies. This was established using a framework involving live cell imaging to measure dynamics, internal rearrangement capacity, coalescence, and relaxation time. Additionally, we integrated thermodynamic characteristics by analysing fixed images through Z-projections. The aforementioned paper laid the foundation for this subsequent technical paper, which explores how different modalities in data acquisition and processing impact the robustness of results to detect bona fide phase transitions by measuring thermodynamic traits in fixed cells. Using solely this approach would greatly simplify screening pipelines. For this, we tested how single focal plane images, Z-projections, or volumetric analyses of images stained with antibodies or live tagged proteins altered the quantification of thermodynamic measurements. Customizing methodologies for different biomolecular condensates through advanced bioimaging significantly contributes to biological research and potential therapeutic advancements.
Assuntos
Vírus da Influenza A , Condensados Biomoleculares , Processamento de Imagem Assistida por Computador , Anticorpos , BiofísicaRESUMO
Introduction: Auditory verbal hallucinations (AVH) are a cardinal symptom of schizophrenia but are also reported in the general population without need for psychiatric care. Previous evidence suggests that AVH may reflect an imbalance of prior expectation and sensory information, and that altered salience processing is characteristic of both psychotic and non-clinical voice hearers. However, it remains to be shown how such an imbalance affects the categorisation of vocal emotions in perceptual ambiguity.Methods: Neutral and emotional nonverbal vocalisations were morphed along two continua differing in valence (anger; pleasure), each including 11 morphing steps at intervals of 10%. College students (N = 234) differing in AVH proneness (measured with the Launay-Slade Hallucination Scale) evaluated the emotional quality of the vocalisations.Results: Increased AVH proneness was associated with more frequent categorisation of ambiguous vocalisations as 'neutral', irrespective of valence. Similarly, the perceptual boundary for emotional classification was shifted by AVH proneness: participants needed more emotional information to categorise a voice as emotional.Conclusions: These findings suggest that emotional salience in vocalisations is dampened as a function of increased AVH proneness. This could be related to changes in the acoustic representations of emotions or reflect top-down expectations of less salient information in the social environment.
Assuntos
Esquizofrenia , Voz , Ira , Emoções , Alucinações/psicologia , HumanosRESUMO
Drug repositioning has been an important ally in the search for new antifungal drugs. Statins are drugs that act to prevent sterol synthesis in both humans and fungi and for this reason they are promissory candidates to be repositioned to treat mycoses. In this study we evaluated the antifungal activity of atorvastatin by in vitro tests to determine the minimum inhibitory concentration against azole resistant Candida albicans and its mechanisms of action. Moreover, the efficacy of both atorvastatin-loaded oral and vaginal emulgels (0.75%, 1.5% and 3% w/w) was evaluated by means of in vivo experimental models of oral and vulvovaginal candidiasis, respectively. The results showed that atorvastatin minimal inhibitory concentration against C. albicans was 31.25 µg/ml. In oral candidiasis experiments, the group treated with oral emulgel containing 3.0% atorvastatin showcased total reduction in fungal load after nine days of treatment. Intravaginal delivery atorvastatin emulgel showed considerable effectiveness at the concentration of 3% (65% of fungal burden reduction) after nine days of treatment. From these findings, it is possible to assert that atorvastatin may be promising for drug repositioning towards the treatment of these opportunistic mycoses.
Atorvastatin is a statin drug that presents antifungal activity. This study showed that atorvastatin-containing oral and vaginal emulgels were able to treat vulvovaginal and oral candidiasis of infected animal model. Therefore, we showcased that atorvastatin may be a possible therapeutic agent in order to be a used to control opportunistic mucosal fungal infections caused by Candida albicans.
Assuntos
Atorvastatina/farmacologia , Atorvastatina/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase Bucal/tratamento farmacológico , Candidíase Vulvovaginal/tratamento farmacológico , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Azóis/farmacologia , Candidíase Bucal/microbiologia , Candidíase Vulvovaginal/microbiologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Farmacorresistência Fúngica , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Ratos , Resultado do TratamentoRESUMO
Mechanisms of viral oncogenesis are diverse and include the off-target activity of enzymes expressed by the infected cells, which evolved to target viral genomes for controlling their infection. Among these enzymes, the single-strand DNA editing capability of APOBECs represent a well-conserved viral infection response that can also cause untoward mutations in the host DNA. Here we show, after evaluating somatic single-nucleotide variations and transcriptome data in 240 gastric cancer samples, a positive correlation between APOBEC3s mRNA-expression and the APOBEC-mutation signature, both increased in EBV+ tumors. The correlation was reinforced by the observation of APOBEC mutations preferentially occurring in the genomic loci of the most active transcripts. This EBV infection and APOBEC3 mutation-signature axis were confirmed in a validation cohort of 112 gastric cancer patients. Our findings suggest that APOBEC3 upregulation in EBV+ cancer may boost the mutation load, providing further clues to the mechanisms of EBV-induced gastric carcinogenesis. After further validation, this EBV-APOBEC axis may prove to be a secondary driving force in the mutational evolution of EBV+ gastric tumors, whose consequences in terms of prognosis and treatment implications should be vetted.
Assuntos
Citidina Desaminase/genética , DNA de Neoplasias/genética , Herpesvirus Humano 4/patogenicidade , Neoplasias Gástricas/virologia , Desaminases APOBEC , Carcinogênese , Genes Virais , Herpesvirus Humano 4/genética , Humanos , Mutação , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Breast cancer (BC) is a major health concern and better understanding of its biology might improve treatment decisions and patient outcomes. Histone3 Lysine27 tri-methylation (H3K27me3) is a post-translational histone modification frequently associated with altered gene expression. In BC patients, lower H3K27me3 expression has been associated with worse prognosis. We assessed H3K27me3 immunoexpression with digital imaging software assistance, in a cohort of luminal-like BC patients with long-term follow-up time and evaluated its association with clinically relevant endpoints and its clinical usefulness. METHODS: H3K27me3 immunoexpression was assessed, by means of digital-imaging system, in archival tissue samples of 160 luminal A/B-like HER2-negative invasive BC, stages I-III. Survival analysis was performed using Kaplan-Meier and Cox regression. Cases were categorized as 'low' or 'high' expression based on cut-off defined by receiver operating characteristic (ROC) curve analysis. RESULTS: The patient cohort showed a median age of 61-years, with a median follow-up time of 11.7 years. Low H3K27me3 expression (below 85% cut-off) was significantly associated with recurrence, both in univariable (HR = 1.99, 95%CI 1.066-3.724) and multivariable analysis when adjusting for grade and age (HR = 1.89, 95%CI 1.004-3.559). A trend for higher risk of death in low H3K27me3 expression BC was observed (p = 0.069), reaching statistical significance in younger patients (p = 0.021). CONCLUSIONS: H3K27me3 immunoexpression assessed by digital imaging scoring software is an independent prognosis biomarker in luminal-like BC patients and may assist in more individualized adjuvant treatment decisions, thus potentially reducing recurrences after curative-intent treatment, while sparing unnecessary toxicity.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação para Baixo , Histonas/metabolismo , Interpretação de Imagem Assistida por Computador/métodos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Metilação , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Medicina de Precisão , Prognóstico , SoftwareRESUMO
Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.
RESUMO
Whereas cancer patients have benefited from liquid biopsies, the scenario for gastric adenocarcinoma (GAC) is still dismal. We used next-generation deep sequencing of TP53-a highly mutated and informative gene in GAC-to assess mutations in tumor biopsies, plasma (PL) and stomach fluids (gastric wash-GW). We evaluated their potential to reveal tumor-derived mutations, useful for monitoring mutational dynamics at diagnosis, progression and treatment. Exon-capture libraries were constructed from 46 patients including tumor biopsies, GW and PL pre and post-treatment (196 samples), with high vertical coverage >8,000×. At diagnosis, we detected TP53 mutations in 15/46 biopsies (32.6%), 7/46 GW- (15.2%) and 6/46 PL-samples (13%). Biopsies and GW were concordant in 38/46 cases (82.6%) for the presence/absence of mutations and, furthermore, four GW-exclusive mutations were identified, suggesting tumor heterogeneity. Considering the combined analysis of GW and PL, TP53 mutations found in biopsies were also identified in 9/15 (60%) of cases, the highest detection level reported for GAC. Our study indicates that GW could be useful to track DNA alterations, especially if anchored to a comprehensive gene-panel designed for this malignancy.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Mutação/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Análise Mutacional de DNA/métodos , Feminino , Seguimentos , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Estômago/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Influenza A is a rapidly evolving virus that is successful in provoking periodic epidemics and occasional pandemics in humans. Viral assembly is complex as the virus incorporates an eight-partite genome of RNA (in the form of viral ribonucleoproteins, vRNPs), and viral genome assembly - with its implications to public health - is not completely understood. It has previously been reported that vRNPs are transported to the cell surface on Rab11-containing vesicles by using microtubules but, so far, no molecular motor has been assigned to the process. Here, we have identified KIF13A, a member of the kinesin-3 family, as the first molecular motor to efficiently transport vRNP-Rab11 vesicles during infection with influenza A. Depletion of KIF13A resulted in reduced viral titers and less accumulation of vRNPs at the cell surface, without interfering with the levels of other viral proteins at sites of viral assembly. In addition, when overexpressed and following two separate approaches to displace vRNP-Rab11 vesicles, KIF13A increased levels of vRNP at the plasma membrane. Together, our results show that KIF13A plays an important role in the transport of influenza A vRNPs, a crucial step for viral assembly.This article has an associated First Person interview with the first author of the paper.
Assuntos
Vírus da Influenza A/patogenicidade , Cinesinas/metabolismo , Transporte Proteico/fisiologia , Ribonucleoproteínas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Genoma Viral/genética , Humanos , Microtúbulos/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologiaRESUMO
Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.
Assuntos
Galanina/genética , Esclerose Múltipla/genética , Receptor Tipo 2 de Galanina/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular , Feminino , Células HEK293 , Humanos , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética , Adulto JovemRESUMO
BACKGROUND: Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. METHODS: Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. RESULTS: MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. CONCLUSIONS: MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients' monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/sangue , MicroRNAs/genética , Adulto , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Estudos de Coortes , Feminino , Humanos , Biópsia Líquida , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Curva ROCRESUMO
Spinocerebellar ataxia type 2 (SCA2) is caused by an unstable expanded CAG repeat tract (CAGexp) at ATXN2. Although prone to selective forces such as anticipation, SCA2 frequency seems to be stable in populations. Our aim was to estimate reproductive success, segregation patterns, and role of anticipation in SCA2. Adult subjects from families with molecular diagnosis provided data about all his/her relatives. Affected and unaffected sibs older than 65.7 years of age were used to estimate reproductive success and segregation patterns. Twenty-one SCA2 families were studied, including 1017 individuals (164 affected) who were born from 1840 to 2012. The median number of children of the non-carriers and carriers, among 99 subjects included in the reproductive success analysis, were 2 and 3 (p < 0.025), respectively. Therefore, the reproductive success of carriers was 1.5. There were 137 non-carriers (59.6%) and 93 carriers (40.4%) (p = 0.04), among subjects included in the segregation analysis. Age at onset across generations pointed to anticipation as a frequent phenomenon. We raised evidence in favor of increased reproductive success related to the carrier state at ATXN2, and segregation distortion favoring normal alleles. Since majority of normal alleles analyzed carried 22 repeats, we propose that this distortion segregation can be related to the high frequency of this allele in human chromosomes.
Assuntos
Aptidão Genética , Seleção Genética , Ataxias Espinocerebelares/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Ataxina-2/genética , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Irmãos , Ataxias Espinocerebelares/epidemiologia , Ataxias Espinocerebelares/fisiopatologia , Expansão das Repetições de Trinucleotídeos , Adulto JovemRESUMO
The original version of this article unfortunately contained a mistake. The spelling of the surname of one co-author from the publication entitled "Selective Forces Related to Spinocerebellar Ataxia Type 2" that was recently published in the journal "The Cerebelum" was incorrect.