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Exploring the potential lead compounds for Alzheimer's disease (AD) remains one of the challenging tasks. Here, we report that the plant extract conophylline (CNP) impeded amyloidogenesis by preferentially inhibiting BACE1 translation via the 5' untranslated region (5'UTR) and rescued cognitive decline in an animal model of APP/PS1 mice. ADP-ribosylation factor-like protein 6-interacting protein 1 (ARL6IP1) was then found to mediate the effect of CNP on BACE1 translation, amyloidogenesis, glial activation, and cognitive function. Through analysis of the 5'UTR-targetd RNA-binding proteins by RNA pulldown combined with LC-MS/MS, we found that FMR1 autosomal homolog 1 (FXR1) interacted with ARL6IP1 and mediated CNP-induced reduction of BACE1 by regulating the 5'UTR activity. Without altering the protein levels of ARL6IP1 and FXR1, CNP treatment promoted ARL6IP1 interaction with FXR1 and inhibited FXR1 binding to the 5'UTR both in vitro and in vivo. Collectively, CNP exhibited a therapeutic potential for AD via ARL6IP1. Through pharmacological manipulation, we uncovered a dynamic interaction between FXR1 and the 5'UTR in translational control of BACE1, adding to the understanding of the pathophysiology of AD.
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Enfermedad de Alzheimer , Animales , Ratones , Regiones no Traducidas 5' , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cromatografía Liquida , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Biosíntesis de Proteínas , Espectrometría de Masas en TándemRESUMEN
AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.
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Subunidades sigma de Complejo de Proteína Adaptadora , Enfermedad de Alzheimer , Ratones , Humanos , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
AIM: To evaluate the effects of bariatric arterial embolization (BAE) on gastric emptying of, and the glycaemic response to, an oral glucose load in an obese canine model with impaired glucose tolerance. METHODS: Eleven male dogs were fed a high-fat, high-fructose diet for 7 weeks before receiving BAE, which involved selective embolization of the left gastric artery (n = 5; 14.9 ± 0.8 kg), or the sham (n = 6; 12.6 ± 0.8 kg) procedure. Postprocedural body weight was measured weekly for 4 weeks. Prior to and at 4 weeks postprocedure, a glucose solution containing 13C-acetate was administered orally for evaluation of the gastric half-emptying time (T50) and the glycaemic response. The relationship between the changes in the blood glucose area under the curve over the first 60 minutes (AUC0-60min) and the T50 was also assessed. RESULTS: At 4 weeks postprocedure, BAE reduced body weight (BAE vs. the sham procedure: -5.7% ± 0.9% vs. 3.5% ± 0.9%, P < .001), slowed gastric emptying (T50 at baseline vs. postprocedure: 75.5 ± 2.0 vs. 82.5 ± 1.8 minutes, P = .021 in the BAE group; 73.8 ± 1.8 vs. 74.3 ± 1.9 minutes in the sham group) and lowered the glycaemic response to oral glucose (AUC0-60min at baseline vs. postprocedure: 99.2 ± 13.7 vs. 67.6 ± 9.8 mmol·min/L, P = .043 in the BAE group; 100.2 ± 13.4 vs. 103.9 ± 14.6 mmol·min/L in the sham group). The change in the glucose AUC0-60min correlated inversely with that of the T50 (r = -0.711; P = .014). CONCLUSIONS: In a canine model with impaired glucose tolerance, BAE, while reducing body weight, slowed gastric emptying and attenuated the glycaemic response to an oral glucose load.
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OBJECTIVES: This study aimed to investigate the efficacy and safety of transarterial chemoembolization (TACE) plus camrelizumab, a monoclonal antibody targeting programmed death-1, and apatinib for patients with intermediate and advanced hepatocellular carcinoma (HCC) in a real-world setting. METHODS: A total of 586 HCC patients treated with either TACE plus camrelizumab and apatinib (combination group, n = 107) or TACE monotherapy (monotherapy group, n = 479) were included retrospectively. Propensity score matching analysis was used to match patients. The overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and safety in the combination group were described in comparison to monotherapy. RESULTS: After propensity score matching (1:2), 84 patients in the combination group were matched to 147 patients in the monotherapy group. The median age was 57 years and 71/84 (84.5%) patients were male in the combination group, while the median age was 57 years with 127/147 (86.4%) male in the monotherapy group. The median OS, PFS, and ORR in the combination group were significantly higher than those in the monotherapy group (median OS, 24.1 vs. 15.7 months, p = 0.008; median PFS, 13.5 vs. 7.7 months, p = 0.003; ORR, 59.5% [50/84] vs. 37.4% [55/147], p = 0.002). On multivariable Cox regression, combination therapy was associated with significantly better OS (adjusted hazard ratio [HR], 0.41; 95% confidence interval [CI], 0.26-0.64; p < 0.001) and PFS (adjusted HR, 0.52; 95% CI, 0.37-0.74; p < 0.001). Grade 3 or 4 adverse events occurred in 14/84 (16.7%) and 12/147 (8.2%) in the combination and monotherapy groups, respectively. CONCLUSIONS: TACE plus camrelizumab and apatinib showed significantly better OS, PFS, and ORR versus TACE monotherapy for predominantly advanced HCC. CLINICAL RELEVANCE STATEMENT: Compared with TACE monotherapy, TACE plus immunotherapy and molecular targeted therapy showed better clinical efficacy for predominantly advanced HCC patients, with a higher incidence of adverse events. KEY POINTS: ⢠This propensity score-matched study demonstrates that TACE plus immunotherapy and molecular targeted therapy have a longer OS, PFS, and ORR compared with TACE monotherapy in HCC. ⢠Grade 3 or 4 adverse events occurred in 14/84 (16.7%) patients treated with TACE plus immunotherapy and molecular targeted therapy compared with 12/147 (8.2%) patients in the monotherapy group, while no grade 5 adverse events were observed in all cohorts.
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Antineoplásicos , Carcinoma Hepatocelular , Quimioembolización Terapéutica , Neoplasias Hepáticas , Humanos , Masculino , Persona de Mediana Edad , Femenino , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Antineoplásicos/uso terapéutico , Quimioembolización Terapéutica/efectos adversos , Puntaje de Propensión , Estudios RetrospectivosRESUMEN
WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.
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Nicotiana , Transcriptoma , Nicotiana/genética , Nicotiana/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , LípidosRESUMEN
Thioredoxin-2 (TXN2) is a mitochondrial protein and represents one of the intrinsic antioxidant enzymes. It has long been recognized that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Alzheimer's disease (AD). We hypothesized that mitochondrial TXN2 might play a role in AD-like pathology. In this study, we found that in SH-SY5Y and HEK cells stably express full-length human amyloid-ß precursor protein (HEK-APP), TXN2 silencing or over-expression selectively increased or decreased the transcription of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), respectively, without altering the protein levels of others enzymes involved in the catalytic processing of APP. As a result, ß-amyloid protein (Aß) levels were significantly decreased by TXN2. In addition, in cells treated with 3-nitropropionic acid (3-NP) that is known to increase reactive oxygen species (ROS) and promote mitochondrial dysfunction, TXN2 silencing resulted in further enhancement of BACE1 protein levels, suggesting a role of TXN2 in ROS removal. The downstream signaling might involve NFκB, as TXN2 reduced the phosphorylation of p65 and IκBα; and p65 knockdown significantly attenuated TXN2-mediated regulation of BACE1. Concomitantly, the levels of cellular ROS, apoptosis-related proteins and cell viability were altered by TXN2 silencing or over-expression. In APPswe/PS1E9 mice, an animal model of AD, the cortical and hippocampal TXN2 protein levels were decreased at 12 months but not at 6 months, suggesting an age-dependent decline. Collectively, TXN2 regulated BACE1 expression and amyloidogenesis via cellular ROS and NFκB signaling. TXN2 might serve as a potential target especially for early intervention of AD.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Mitocondriales/metabolismo , Tiorredoxinas/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Mitocondrias/metabolismo , Transducción de Señal/fisiologíaRESUMEN
MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs that play important roles in abiotic stress responses in plants, but their regulatory roles in the adaptive response to heat stress at the booting stage in two rice varieties, 9311 and Nagina 22, remain largely unknown. In this study, 464 known miRNAs and 123 potential novel miRNAs were identified. Of these miRNAs, a total of 90 differentially expressed miRNAs were obtained with 9311 libraries as the control group, of which 54 were upregulated and 36 were downregulated. To gain insight into functional significance, 2773 potential target genes of these 90 differentially expressed miRNAs were predicted. GO enrichment analysis showed that the predicted target genes of differentially expressed miRNAs included NACs, LACs, CSD, and Hsp40. KEGG pathway analysis showed that the target genes of these differentially expressed miRNAs were significantly enriched in the plant hormone signal transduction pathway. The expression levels of 10 differentially expressed miRNAs and their target genes obtained by qRT-PCR were largely consistent with the sequencing results. This study lays a foundation for the elucidation of the miRNA-mediated regulatory mechanisms in rice at elevated temperatures.
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MicroARNs , Oryza , Estrés Fisiológico , Temperatura , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/genética , Oryza/genética , ARN de Planta/genética , Análisis de Secuencia de ARNRESUMEN
Renal fibrosis is common especially in the elderly population. Recently, we found that vitamin D deficiency caused prostatic hyperplasia. This study aimed to investigate whether vitamin D deficiency promotes renal fibrosis and functional impairment. All mice except controls were fed with vitamin D-deficient (VDD) diets, beginning from their early life. The absolute and relative kidney weights on postnatal week 20 were decreased in VDD diet-fed male pups but not in female pups. A mild pathological damage was observed in VDD diet-fed male pups but not in females. Further analysis showed that VDD-induced pathological damage was aggravated, accompanied by renal dysfunction in 40-week-old male pups. An obvious collagen deposition was observed in VDD diet-fed 40-week-old male pups. Moreover, renal α-smooth muscle actin (α-SMA), a marker of epithelial-mesenchymal transition (EMT), and Tgf-ß mRNA were up-regulated. The in vitro experiment showed that 1,25-dihydroxyvitamin D3 alleviated transforming growth factor-ß1 (TGF-ß1)-mediated down-regulation of E-cadherin and inhibited TGF-ß1-evoked up-regulation of N-cadherin, vimentin and α-SMA in renal epithelial HK-2 cells. Moreover, 1,25-dihydroxyvitamin D3 suppressed TGF-ß1-evoked Smad2/3 phosphorylation in HK-2 cells. These results provide experimental evidence that long-term vitamin D deficiency promotes renal fibrosis and functional impairment, at least partially, through aggravating TGF-ß/Smad2/3-mediated EMT in middle-aged male mice.
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Enfermedades Renales/etiología , Riñón/patología , Riñón/fisiopatología , Deficiencia de Vitamina D/complicaciones , Actinas/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Calcitriol/farmacología , Línea Celular , Colecalciferol/farmacología , Transición Epitelial-Mesenquimal , Femenino , Fibrosis/etiología , Fibrosis/patología , Humanos , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Tamaño de los Órganos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Vimentina/metabolismo , Vitamina D/análogos & derivados , Vitamina D/sangre , Deficiencia de Vitamina D/patología , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was used to discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.
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Proteínas Argonautas/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Argonautas/metabolismo , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo , Células HEK293 , Células HeLa , Humanos , Fosforilación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia ArribaAsunto(s)
Carcinoma Hepatocelular , Hepatitis B Crónica , Hepatitis B , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B/fisiología , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Hepatitis B Crónica/tratamiento farmacológico , Activación Viral , Hepatitis B/tratamiento farmacológicoRESUMEN
1-Nitropyrene (1-NP), a typical nitrated polycyclic aromatic hydrocarbon, is widely distributed in the environment and is well known for its mutagenic effects. Recently, we found that gestational 1-NP exposure induced fetal growth restriction. In this study, we further evaluated the effect of in utero 1-NP exposure on postnatal growth and neurobehavioral development in the offspring. Pregnant mice were administered with 1-NP (10⯵g/kg) by gavage daily in late pregnancy (GD13-GD17). The body weight of each offspring was measured from PND1 to 12 weeks postpartum. Exploration and anxiety related activities were detected by open-field test at 6 weeks postpartum. Learning and memory were assessed by Morris Water Maze at 7 weeks postpartum. And depressive-like behaviors were estimated by sucrose preference test at 10 weeks postpartum. Significant body weight reduction was observed in 1-NP-exposed female offspring at PND1, PND14 and PND21 while the lower body weight was only found at PND1 for 1-NP-exposed male offspring. Exploration and anxiety activities at puberty, and depressive-like behavior in adulthood were not disturbed in offspring prenatally exposed to 1-NP. Interestingly, spatial learning and memory ability at puberty was impaired in females but not in males prenatally exposed to 1-NP. These findings suggest that gestational 1-NP exposure delays postnatal growth and impaired neurobehavioral development in a gender-dependent manner.
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Contaminantes Ambientales/toxicidad , Exposición Materna/efectos adversos , Mutágenos/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Pirenos/toxicidad , Animales , Femenino , Masculino , Memoria/efectos de los fármacos , Ratones Endogámicos ICR , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/psicología , Factores Sexuales , Aprendizaje Espacial/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
Factor V Leiden and factor II c.*97G>A (formerly referred to as prothrombin 20210G>A) are the two most common genetic variants associated with venous thromboembolism (VTE). Testing for these variants is one of the most common referrals in clinical genetics laboratories. While the methodologies for testing these two variants are relatively straightforward, the clinical implementation can be complicated with regard to test indications, risk assessment of occurrence and recurrence of VTE, and related genetic counseling. This document provides an overview of VTE, information about the variants and their influence on risk, considerations before initiating genetic testing, and the clinical and analytical sensitivity and specificity of the tests. Key information that should be included in the laboratory report is also provided. Disease-specific statements are intended to augment the general American College of Medical Genetics and Genomics (ACMG) technical standards for clinical genetics laboratories. Individual laboratories are responsible for meeting the Clinical Laboratory Improvement Amendments (CLIA)/College of American Pathologists (CAP) quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency testing, and quality control measures. This 2018 edition of the ACMG technical standard updates and supersedes the 2005 edition on this topic. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and the methods of analysis.
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Factor V/genética , Pruebas Genéticas/normas , Genética Médica , Tromboembolia Venosa/diagnóstico , Variación Genética , Genómica , Humanos , Laboratorios/normas , Mutación , Estados Unidos/epidemiología , Tromboembolia Venosa/epidemiología , Tromboembolia Venosa/genéticaRESUMEN
To define the functional pathways regulating epithelial cell migration, we performed a genome-wide RNAi screen using 55,000 pooled lentiviral shRNAs targeting â¼11,000 genes, selecting for transduced cells with increased motility. A stringent validation protocol generated a set of 31 genes representing diverse pathways whose knockdown dramatically enhances cellular migration. Some of these pathways share features of epithelial-to-mesenchymal transition (EMT), and together they implicate key regulators of transcription, cellular signaling, and metabolism, as well as novel modulators of cellular trafficking, such as DLG5. In delineating downstream pathways mediating these migration phenotypes, we observed universal activation of ERKs and a profound dependence on their RSK effectors. Pharmacological inhibition of RSK dramatically suppresses epithelial cell migration induced by knockdown of all 31 genes, suggesting that convergence of diverse migratory pathways on this kinase may provide a therapeutic opportunity in disorders of cell migration, including cancer metastasis.
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Movimiento Celular/genética , Estudio de Asociación del Genoma Completo , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas/metabolismo , Línea Celular Tumoral , Células Epiteliales/citología , Humanos , Proteínas de la Membrana/metabolismo , Mesodermo/citología , Reproducibilidad de los Resultados , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Most gastrointestinal stromal tumors (GISTs) harbor mutually exclusive gain of function mutations in the receptor tyrosine kinase (RTK) KIT (70-80%) or in the related receptor PDGFRA (~10%). These GISTs generally respond well to therapy with the RTK inhibitor imatinib mesylate (IM), although initial response is genotype-dependent. An alternate mechanism leading to GIST oncogenesis is deficiency in the succinate dehydrogenase (SDH) enzyme complex resulting from genetic or epigenetic inactivation of one of the four SDH subunit genes (SDHA, SDHB, SDHC, SDHD, collectively referred to as SDHX). SDH loss of function is generally seen only in GIST lacking RTK mutations, and SDH-deficient GIST respond poorly to imatinib therapy. METHODS: Tumor and normal DNA from a GIST case carrying the IM-resistant PDGFRA D842V mutation was analyzed by whole exome sequencing (WES) to identify additional potential targets for therapy. The tumors analyzed were separate recurrences following progression on imatinib, sunitinib, and the experimental PDGFRA inhibitor crenolanib. Tumor sections from the GIST case and a panel of ~75 additional GISTs were subjected to immunohistochemistry (IHC) for the SDHB subunit. RESULTS: Surprisingly, a somatic, loss of function mutation in exon 4 of the SDHB subunit gene (c.291_292delCT, p.I97Mfs*21) was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and IHC for the SDHB subunit demonstrated that these tumors were SDH-deficient. IHC for the SDHB subunit across a panel of ~75 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations. CONCLUSIONS: This is the first reported case of a PDGFRA mutant GIST exhibiting SDH-deficiency. A brief discussion of the relevant GIST literature is included.
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Tumores del Estroma Gastrointestinal/genética , Mutación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Succinato Deshidrogenasa/deficiencia , Biomarcadores , Análisis Mutacional de ADN , Tumores del Estroma Gastrointestinal/diagnóstico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Humanos , Inmunohistoquímica , Polimorfismo de Nucleótido Simple , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Secuenciación Completa del GenomaRESUMEN
AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5' to 3' LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.
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Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/genética , Edición de ARN/genética , ARN/genética , Latencia del Virus/genética , Secuencia de Bases , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma Humano/genética , Células HEK293 , Infecciones por VIH/inmunología , Duplicado del Terminal Largo de VIH/genética , Humanos , Datos de Secuencia Molecular , Vacunación , ARN Pequeño no TraducidoRESUMEN
BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.
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Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Roturas del ADN de Doble Cadena , Variación Genética , Linfocitos T/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Bases de Datos Genéticas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Exoma , Femenino , Frecuencia de los Genes , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Células HCT116 , Herencia , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutágenos/farmacología , Fenotipo , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Análisis de Secuencia de ADN , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección , Regulación hacia Arriba , Helicasa del Síndrome de WernerRESUMEN
Analysis of the molecular etiologies of SCID has led to important insights into the control of immune cell development. Most cases of SCID result from either X-linked or autosomal recessive inheritance of mutations in a known causative gene. However, in some cases, the molecular etiology remains unclear. To identify the cause of SCID in a patient known to lack the protein-tyrosine phosphatase CD45, we used SNP arrays and whole-exome sequencing. The patient's mother was heterozygous for an inactivating mutation in CD45 but the paternal alleles exhibited no detectable mutations. The patient exhibited a single CD45 mutation identical to the maternal allele. Patient SNP array analysis revealed no change in copy number but loss of heterozygosity for the entire length of chromosome 1 (Chr1), indicating that disease was caused by uniparental disomy (UPD) with isodisomy of the entire maternal Chr1 bearing the mutant CD45 allele. Nonlymphoid blood cells and other mesoderm- and ectoderm-derived tissues retained UPD of the entire maternal Chr1 in this patient, who had undergone successful bone marrow transplantation. Exome sequencing revealed mutations in seven additional genes bearing nonsynonymous SNPs predicted to have deleterious effects. These findings are unique in representing a reported case of SCID caused by UPD and suggest UPD should be considered in SCID and other recessive disorders, especially when the patient appears homozygous for an abnormal gene found in only one parent. Evaluation for alterations in other genes affected by UPD should also be considered in such cases.
Asunto(s)
Antígenos Comunes de Leucocito/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Disomía Uniparental , Heterocigoto , Humanos , Antígenos Comunes de Leucocito/genética , Pérdida de Heterocigocidad , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Animal acariasis is one of the important veterinary skin diseases. Chemical drugs have been widely used to treat and control this kind of disease. But many chemicals control could increase resistance in target species, toxicity and environmental hazards. We found that the 9-oxo-10, 11-dehydroageraphorone (euptox A) extracted from E. adenophorum has strong toxicity against P. cuniculi in vitro, but the in vivo acaricidal actions of euptox A have yet to be investigated. RESULTS: A 14-day experiment was performed using rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups; animals in groups A, B and C were treated in each ear topically with 4.0 ml of 2.0 and 1.0 g/L (w/v) euptox A, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7 and 14, to inspect the presence or absence of mites and scabs/crusts. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.48, 3.37, 3.43 and 3.45 to 0.37, 0.42, 0.78 and 0.38 in the ears of animals in groups A, B , C and D, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not increase significantly (P > 0.05) during the experiment, and this changed from 3.32 to 3.37 in the ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (0 and 7 d), the rabbits in groups A, B, C and D had recovered completely 14 days after the last treatment and no recurrences of infection were observed. CONCLUSIONS: These results indicate that euptox A was potent compounds for the effective control of animal P. cuniculi in vivo.
Asunto(s)
Acaricidas/uso terapéutico , Infestaciones por Ácaros/veterinaria , Psoroptidae/efectos de los fármacos , Sesquiterpenos/uso terapéutico , Acaricidas/aislamiento & purificación , Ageratina/química , Animales , Relación Dosis-Respuesta a Droga , Ivermectina/uso terapéutico , Infestaciones por Ácaros/tratamiento farmacológico , Conejos , Sesquiterpenos/aislamiento & purificación , Resultado del TratamientoRESUMEN
A comprehensive understanding of the molecular vulnerabilities of every type of cancer will provide a powerful roadmap to guide therapeutic approaches. Efforts such as The Cancer Genome Atlas Project will identify genes with aberrant copy number, sequence, or expression in various cancer types, providing a survey of the genes that may have a causal role in cancer. A complementary approach is to perform systematic loss-of-function studies to identify essential genes in particular cancer cell types. We have begun a systematic effort, termed Project Achilles, aimed at identifying genetic vulnerabilities across large numbers of cancer cell lines. Here, we report the assessment of the essentiality of 11,194 genes in 102 human cancer cell lines. We show that the integration of these functional data with information derived from surveying cancer genomes pinpoints known and previously undescribed lineage-specific dependencies across a wide spectrum of cancers. In particular, we found 54 genes that are specifically essential for the proliferation and viability of ovarian cancer cells and also amplified in primary tumors or differentially overexpressed in ovarian cancer cell lines. One such gene, PAX8, is focally amplified in 16% of high-grade serous ovarian cancers and expressed at higher levels in ovarian tumors. Suppression of PAX8 selectively induces apoptotic cell death of ovarian cancer cells. These results identify PAX8 as an ovarian lineage-specific dependency. More generally, these observations demonstrate that the integration of genome-scale functional and structural studies provides an efficient path to identify dependencies of specific cancer types on particular genes and pathways.