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This qualitative study used an artificial intelligence (AI) large language model and social media to investigate challenges encountered by transgender individuals during breast and gynecological cancer care.
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Disparidades en Atención de Salud , Neoplasias , Medios de Comunicación Sociales , Personas Transgénero , Humanos , Medios de Comunicación Sociales/estadística & datos numéricos , Personas Transgénero/estadística & datos numéricos , Personas Transgénero/psicología , Neoplasias/terapia , Femenino , Masculino , Inteligencia ArtificialRESUMEN
PURPOSE: Molnupiravir is an oral antiviral agent authorized for emergency use to treat mild to moderate cases of coronavirus disease 2019 (COVID-19) in adults at high risk for progression to severe clinical outcomes. This study aimed to describe patient characteristics and health outcomes in a cohort of adult patients treated with molnupiravir in an outpatient setting in the United States. METHODS: This was a retrospective cohort study of adults identified in the HealthVerity database with a pharmacy claim for molnupiravir between December 24, 2021, and April 14, 2022. Hospitalization and health care use were assessed over the 28 days after the molnupiravir pharmacy claim. FINDINGS: Among 26,554 patients identified, 71.1% were aged ≥50 years and 58.9% were female. A total of 8794 patients (33.1%) had received at least 1 dose of the COVID-19 vaccine. The most prevalent risk factors for severe COVID-19 identified were hypertension (45.1%), steroid and/or immunosuppressant use (39.6%), and being obese or overweight (24.6%), with 79.1% of patients having ≥1 risk factor. The majority (61.0%) of patients were prescribed comedications contraindicated with or had the potential for major drug-drug interactions with ritonavir-containing regimens. Within 28 days after initiating molnupiravir, 3.3% of patients were hospitalized for any cause and 1.7% for COVID-19-related reasons. Among all hospitalized patients, 9.2% were admitted to an intensive care unit, 3.9% received oxygen, and 3.8% required mechanical ventilation. IMPLICATIONS: The majority of patients treated with molnupiravir in early 2022 had at least one risk factor for severe COVID-19 and had comedications that could require treatment modification or monitoring if given a ritonavir-containing regimen. Hospitalization was uncommon after treatment with molnupiravir, with COVID-19-related inpatient admission in <2% of patients. Among those hospitalized, patient use of intensive care and oxygen-based resources was infrequent. The study design, however, does not permit any conclusions regarding the effectiveness of molnupiravir.
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Vacunas contra la COVID-19 , COVID-19 , Adulto , Humanos , Femenino , Masculino , Estudios Retrospectivos , Ritonavir/uso terapéutico , Pacientes Internos , Hospitalización , COVID-19/epidemiología , OxígenoRESUMEN
This study investigates the use of deep learning methods to improve the accuracy of a predictive model for dementia, and compares the performance to a traditional machine learning model. With sufficient accuracy the model can be deployed as a first round screening tool for clinical follow-up including neurological examination, neuropsychological testing, imaging and recruitment to clinical trials. Seven cohorts with two years of data, three to eight years prior to index date, and an incident cohort were created. Four trained models for each cohort, boosted trees, feed forward network, recurrent neural network and recurrent neural network with pre-trained weights, were constructed and their performance compared using validation and test data. The incident model had an AUC of 94.4% and F1 score of 54.1%. Eight years removed from index date the AUC and F1 scores were 80.7% and 25.6%, respectively. The results for the remaining cohorts were between these ranges. Deep learning models can result in significant improvement in performance but come at a cost in terms of run times and hardware requirements. The results of the model at index date indicate that this modeling can be effective at stratifying patients at risk of dementia. At this time, the inability to sustain this quality at longer lead times is more an issue of data availability and quality rather than one of algorithm choices.
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Demencia/diagnóstico , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Aprendizaje Profundo , Demencia/epidemiología , Registros Electrónicos de Salud , Femenino , Humanos , Masculino , Redes Neurales de la Computación , Factores de RiesgoRESUMEN
AIM: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. MATERIALS & METHODS: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. RESULTS: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. CONCLUSION: These data support the strategy and the potential use of the biomarkers for PrCa.
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The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Nefropatías Diabéticas/metabolismo , Endotelina-1/metabolismo , Endotelio/metabolismo , Glomérulos Renales/metabolismo , Mitocondrias/metabolismo , Receptor de Endotelina A/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Anciano , Albuminuria , Animales , Antioxidantes/farmacología , Cromatografía Líquida de Alta Presión , ADN Mitocondrial/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Susceptibilidad a Enfermedades , Endotelio/efectos de los fármacos , Endotelio/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Masculino , Células Mesangiales/patología , Células Mesangiales/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/farmacología , Consumo de Oxígeno , Piperidinas/farmacología , Podocitos/patología , Podocitos/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Adulto JovenRESUMEN
BACKGROUND: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. RESULTS: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. CONCLUSIONS: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.
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Diatomeas/metabolismo , Código de Histonas , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Cromatina/metabolismo , Diatomeas/genética , Expresión Génica , Genómica , Espectrometría de Masas , Nitratos/metabolismo , Nucleosomas/metabolismoRESUMEN
BACKGROUND: Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. RESULTS: We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse "exceptions" from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin. CONCLUSIONS: These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.
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Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Mapeo Cromosómico , Drosophila/genética , Genoma de los Insectos , Animales , Sitios de Unión , Cromatina/metabolismo , Biología Computacional/métodos , Desoxirribonucleasa I/metabolismo , Unión ProteicaRESUMEN
Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1-mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.
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Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Gluconeogénesis/genética , Hígado/metabolismo , Factores de Transcripción/metabolismo , Animales , Células COS , Chlorocebus aethiops , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/genética , Ingestión de Alimentos/fisiología , Ayuno/metabolismo , Glucosa/metabolismo , Células Hep G2 , Homeostasis/fisiología , Humanos , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Current histopathological classification and TNM staging have limited accuracy in predicting survival and stratifying patients for appropriate treatment. The goal of the study is to determine whether the expression pattern of functionally important regulatory proteins can add additional values for more accurate classification and prognostication of non-small lung cancer (NSCLC). METHODS: The expression of 108 proteins and phosphoproteins in 30 paired NSCLC samples were assessed using Protein Pathway Array (PPA). The differentially expressed proteins were further confirmed using a tissue microarray (TMA) containing 94 NSCLC samples and were correlated with clinical data and survival. RESULTS: Twelve of 108 proteins (p-CREB(Ser133), p-ERK1/2(Thr202/Tyr204), Cyclin B1, p-PDK1(Ser241), CDK4, CDK2, HSP90, CDC2p34, ß-catenin, EGFR, XIAP and PCNA) were selected to build the predictor to classify normal and tumor samples with 97% accuracy. Five proteins (CDC2p34, HSP90, XIAP, CDK4 and CREB) were confirmed to be differentially expressed between NSCLC (n=94) and benign lung tumor (n=19). Over-expression of CDK4 and HSP90 in tumors correlated with a favorable overall survival in all NSCLC patients and the over-expression of p-CREB(Ser133) and CREB in NSCLC correlated with a favorable survival in smokers and those with squamous cell carcinoma, respectively. Finally, the four proteins (CDK4, HSP90, p-CREB and CREB) were used to calculate the risk score of each individual patient with NSCLC to predict survival. CONCLUSION: In summary, our data demonstrated a broad disturbance of functionally important regulatory proteins in NSCLC and some of these can be selected as clinically useful biomarkers for diagnosis, classification and prognosis.
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We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness). Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.
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Chlamydia trachomatis/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Metagenoma , Transcriptoma , Línea Celular Tumoral , Chlamydia trachomatis/metabolismo , Colágeno/genética , Colágeno/metabolismo , Células Epiteliales/microbiología , Retroalimentación Fisiológica , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Análisis de Secuencia de ARN , Tenascina/genética , Tenascina/metabolismoRESUMEN
The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0 fold following CBDL. The most promising candidate genes included MUPs, Serpina1a and LCN-2. RT-PCR validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP-1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate inter-individual variability similar to what we have shown in mice, then a "personalized medicine" approach to cholestatic patients may become possible.
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Helicobacter pylori (H. pylori) infects the gastric mucosa and persists for the life of the host. Bacterial persistence may be due to the induction of regulatory T cells (Tregs) whichmay have protective effects against other diseases such as asthma. It has been shown that H. pylori modulates the T cell response through dendritic cell reprogramming but the molecular pathways involved are relatively unknown. The goal of this study was to identify critical elements of dendritic cell (DC) activation and evaluate potential influence on immune activation. Microarray analysis was used to demonstrate limited gene expression changes in H. pylori stimulated bone marrow derived DCs (BMDCs) compared to the BMDCs stimulated with E. coli. IRAK-M, a negative regulator of TLR signaling, was upregulated and we selectedit for investigation of its role in modulating the DC and T cell responses. IRAK-M(-/-) and wild type BMDC were compared for their response to H. pylori. Cells lacking IRAK-M produced significantly greater amounts of proinflammatory MIP-2 and reduced amounts of immunomodulatory IL-10 than wild type BMDC. IRAK-M(-/-) cells also demonstrated increased MHC II expression upon activation. However, IRAK-M(-/-) BMDCs were comparable to wild type BMDCs in inducing T-helper 17 (TH17) and Treg responses as demonstrated in vitro using BMDC CD4+ T cells co-culture assays,and in vivo though the adoptive transfer of CD4(+) FoxP3-GFP T cells into H. pylori infected IRAK-M(-/-) mice. These results suggest that H. pylori infection leads to the upregulation of anti-inflammatory molecules like IRAK-M and that IRAK-M has a direct impact on innate functions in DCs such as cytokine and costimulation molecule upregulation but may not affect T cell skewing.
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Citocinas/metabolismo , Células Dendríticas/metabolismo , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Animales , Quimiocina CXCL2/metabolismo , Helicobacter pylori/patogenicidad , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-10/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid ß-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment.
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Cardiomiopatías/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Cardiomiopatías/genética , Línea Celular , Cricetinae , ADN Mitocondrial/genética , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Perilipina-5 , Proteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Triglicéridos/metabolismoRESUMEN
Throughout infection, pathogenic bacteria induce dramatic changes in host transcriptional repertoires. An understanding of how bacterial factors influence host reprogramming will provide insight into disease pathogenesis. In the human respiratory pathogen Bordetella pertussis, the causative agent of whooping cough, pertussis toxin (PT) is a key virulence factor that promotes colonization, suppresses innate immune responses during early infection, and causes systemic disease symptoms. To determine the full extent of PT-associated gene regulation in the airways through the peak of infection, we measured global transcriptional profiles in the lungs of BALB/c mice infected with wild-type (WT) or PT-deficient (ΔPT) B. pertussis. ΔPT bacteria were inoculated at a dose equivalent to the WT dose and at a high dose (ΔPT(high)) to distinguish effects caused by higher bacterial loads achieved in WT infection from effects associated with PT. The results demonstrated that PT was associated with a significant upregulation of immune and inflammatory response genes as well as several other genes implicated in airway pathology. In contrast to the early, transient responses observed for ΔPT(high) infection, WT infection induced a prolonged expression of inflammatory genes and increased the extent and duration of lung histopathology. In addition, the administration of purified PT to ΔPT(high)-infected mice 1 day after bacterial inoculation exacerbated and prolonged inflammatory responses and airway pathology. These data indicate that PT not only is associated with exacerbated host airway responses during peak B. pertussis infection but also may inhibit host mechanisms of attenuating and resolving inflammation in the airways, suggesting possible links between PT and pertussis disease symptoms.
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Infecciones por Bordetella/fisiopatología , Bordetella pertussis/patogenicidad , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inmunología , Pulmón/inmunología , Toxina del Pertussis/inmunología , Tos Ferina/fisiopatología , Animales , Infecciones por Bordetella/inmunología , Infecciones por Bordetella/metabolismo , Infecciones por Bordetella/patología , Bordetella pertussis/inmunología , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Toxina del Pertussis/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Tos Ferina/inmunología , Tos Ferina/microbiología , Tos Ferina/patologíaRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca(2+)) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase-dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca(2+) influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca(2+) influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca(2+) influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS-related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca(2+) and ROS signaling in DMD and could be effective therapeutic targets for intervention.
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Microtúbulos/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Adolescente , Adulto , Animales , Western Blotting , Calcio/metabolismo , Niño , Preescolar , Humanos , Inmunohistoquímica , Técnicas In Vitro , Lactante , Masculino , Ratones , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , NADPH Oxidasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética , Moduladores de Tubulina/farmacologíaRESUMEN
BACKGROUND: Numerous methods exist for enriching bacterial or mammalian mRNA prior to transcriptome experiments. Yet there persists a need for methods to enrich for mRNA in non-mammalian animal systems. For example, insects contain many important and interesting obligate intracellular bacteria, including endosymbionts and vector-borne pathogens. Such obligate intracellular bacteria are difficult to study by traditional methods. Therefore, genomics has greatly increased our understanding of these bacteria. Efficient subtraction methods are needed for removing both bacteria and insect rRNA in these systems to enable transcriptome-based studies. FINDINGS: A method is described that efficiently removes >95% of insect rRNA from total RNA samples, as determined by microfluidics and transcriptome sequencing. This subtraction yielded a 6.2-fold increase in mRNA abundance. Such a host rRNA-depletion strategy, in combination with bacterial rRNA depletion, is necessary to analyze transcription of obligate intracellular bacteria. Here, transcripts were identified that arise from a lateral gene transfer of an entire Wolbachia bacterial genome into a Drosophila ananassae chromosome. In this case, an rRNA depletion strategy is preferred over polyA-based enrichment since transcripts arising from bacteria-to-animal lateral gene transfer may not be poly-adenylated. CONCLUSIONS: This enrichment method yields a significant increase in mRNA abundance when poly-A selection is not suitable. It can be used in combination with bacterial rRNA subtraction to enable experiments to simultaneously measure bacteria and insect mRNA in vector and endosymbiont biology experiments.
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Drosophila/genética , Drosophila/microbiología , Perfilación de la Expresión Génica/métodos , Transferencia de Gen Horizontal/genética , ARN Ribosómico 18S/aislamiento & purificación , Wolbachia/genética , Actinas/genética , Animales , Genes de Insecto/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genéticaRESUMEN
Current methods have limited accuracy in predicting survival and stratifying patients with gastric cancer for appropriate treatment. We sought to identify protein signatures of gastric cancer for classification and prognostication. The Protein Pathway Array (initial study) and Western blot (confirmation) were used to assess the protein expression in a total of 199 fresh frozen gastric samples. There were 56 paired samples divided into a training set (n = 37) and a validation set (n = 19) for the identification of differentially expressed proteins between tumor and normal tissues. There were 56 tumor samples used to identify proteins correlating with tumor and nodal staging. All 93 tumor samples were used to identify candidate proteins for predicting survival. We confirmed the survival prediction of the candidate proteins by using an additional cohort of gastric cancer samples (n = 50). There were 22 proteins differentially expressed between normal and tumor tissues. Nine proteins were selected to build the predictor to classify normal and tumor samples. Ten proteins were differentially expressed among different T stages and four of these were associated with invasive behavior. An additional four proteins were associated with lymph node metastasis. Two proteins were identified as independent risk factors for overall survival. This study indicated that some dysregulated signaling proteins could be selected as useful biomarkers for tumor classification and predicting outcome in gastric cancer patients.
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Proteínas de Neoplasias/metabolismo , Proteómica/métodos , Transducción de Señal , Neoplasias Gástricas/clasificación , Neoplasias Gástricas/metabolismo , Adulto , Autorradiografía , Análisis por Conglomerados , Demografía , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Fosforilación , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Riesgo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologíaRESUMEN
Our objective was to analyze gene expression pattern in muscles from patients with amyotrophic lateral sclerosis (ALS) and multifocal motor neuropathy (MMN) compared to controls. Biopsied skeletal muscles from three ALS, three MMN and three control subjects had total RNA extracted and subjected to genome-wide gene expression analysis using Affymetrix GeneChip Exon 1.0 ST array. The most significant expression pattern differences were confirmed with RT-PCR in four additional ALS patients. Results showed that over 3000 genes were identified across the groups using q < 10%. Among 50 genes that were overexpressed only in the ALS group were: leucine-rich repeat kinase-2, follistatin, collagen type XIX alpha-1, ceramide kinase-like, sestrin-3 and CXorf64. No genes were significantly overexpressed in MMN alone. Underexpressed genes only in ALS included actinin α3, fructose-1,6-bisphosphatase-2 and homeobox C10; whereas only in MMN: hemoglobin A1 and CXorf64. Ankyrin repeat domain-1 was overexpressed in both groups. Underexpressed genes in both groups included myosin light chain kinase-2, enolase-3 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-1. Validation analysis using RT-PCR confirmed the data for leucine-rich repeat kinase-2, follistatin, collagen type XIX alpha-1, ceramide kinase-like, sestrin-3 and CXorf64. In conclusion, there is differential tissue-specific gene expression in patients with ALS relative to MMN and controls. Further studies are necessary to evaluate the identified genes in larger patient groups and different tissues.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expresión Génica , Proteínas Musculares/genética , Adulto , Anciano , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND/AIM: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression. METHODS/RESULTS: Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling. CONCLUSION: This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role of SERCA2a normalization in multiple pathways in diabetic cardiomyopathy.
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Diabetes Mellitus Tipo 2/genética , Cardiopatías/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Animales , Western Blotting , Diabetes Mellitus Tipo 2/complicaciones , Perfilación de la Expresión Génica , Cardiopatías/complicaciones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Long-Evans , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , TransfecciónRESUMEN
Dicer is an enzyme that generates microRNA (miRNA), which are small, noncoding RNA that function as important regulators of gene and protein expression. For exploration of the functional roles of miRNA in glomerular biology, Dicer was inactivated selectively in mouse podocytes. Mutant mice developed proteinuria 4 to 5 weeks after birth and died several weeks later, presumably from kidney failure. Multiple abnormalities were observed in glomeruli of mutant mice, including foot process effacement, irregular and split areas of the glomerular basement membrane, podocyte apoptosis and depletion, mesangial expansion, capillary dilation, and glomerulosclerosis. Gene profiling revealed upregulation of 190 genes in glomeruli isolated from mutant mice at the onset of proteinuria compared with control littermates. Target sequences for 16 miRNA were significantly enriched in the 3'-untranslated regions of the 190 upregulated genes. Further suggesting validity of the in silico analysis, six of the eight top-candidate miRNA were identified in miRNA libraries generated from podocyte cultures; these included four members of the mir-30 miRNA family, which are known to degrade target transcripts directly. Among 15 upregulated target genes of the mir-30 miRNA, four genes known to be expressed and/or functional in podocytes were identified, including receptor for advanced glycation end product, vimentin, heat-shock protein 20, and immediate early response 3. Receptor for advanced glycation end product and immediate early response 3 are known to mediate podocyte apoptosis, whereas vimentin and heat-shock protein-20 are involved in cytoskeletal structure. Taken together, these results provide a knowledge base for ongoing investigations to validate functional roles for the mir-30 miRNA family in podocyte homeostasis and podocytopathies.