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The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSCderived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3aregulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.
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Neoplasias Óseas , Carcinogénesis , Factor de Transcripción E2F3 , Regulación Neoplásica de la Expresión Génica , Células Madre Pluripotentes Inducidas , Osteosarcoma , Proteínas de Unión a Retinoblastoma , Empalmosomas , Ubiquitina-Proteína Ligasas , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Carcinogénesis/genética , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Genes de Retinoblastoma , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Osteosarcoma/genética , Osteosarcoma/patología , Neoplasias de la Retina/genética , Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Turner syndrome (TS) is a genetic condition occurring in ~1 in 2000 females characterized by the complete or partial absence of the second sex chromosome. TS research faces similar challenges to many other pediatric rare disease conditions, with homogenous, single-center, underpowered studies. Secondary data analyses utilizing electronic health record (EHR) have the potential to address these limitations; however, an algorithm to accurately identify TS cases in EHR data is needed. We developed a computable phenotype to identify patients with TS using PEDSnet, a pediatric research network. This computable phenotype was validated through chart review; true positives and negatives and false positives and negatives were used to assess accuracy at both primary and external validation sites. The optimal algorithm consisted of the following criteria: female sex, ≥1 outpatient encounter, and ≥3 encounters with a diagnosis code that maps to TS, yielding an average sensitivity of 0.97, specificity of 0.88, and C-statistic of 0.93 across all sites. The accuracy of any estradiol prescriptions yielded an average C-statistic of 0.91 across sites and 0.80 for transdermal and oral formulations separately. PEDSnet and computable phenotyping are powerful tools in providing large, diverse samples to pragmatically study rare pediatric conditions like TS.
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Registros Electrónicos de Salud , Síndrome de Turner , Humanos , Niño , Femenino , Síndrome de Turner/diagnóstico , Síndrome de Turner/genética , Fenotipo , Algoritmos , EstradiolRESUMEN
While digital tools, such as the Internet, smartphones, and social media, are an important part of modern society, little is known about the specific role they play in the healthcare management of individuals and caregivers affected by rare disease. Collectively, rare diseases directly affect up to 10% of the global population, suggesting that a significant number of individuals might benefit from the use of digital tools. The purpose of this qualitative interview-based study was to explore: (a) the ways in which digital tools help the rare disease community; (b) the healthcare gaps not addressed by current digital tools; and (c) recommended digital tool features. Individuals and caregivers affected by rare disease who were comfortable using a smartphone and at least 18 years old were eligible to participate. We recruited from rare disease organizations using purposive sampling in order to achieve a diverse and information rich sample. Interviews took place over Zoom and reflexive thematic analysis was utilized to conceptualize themes. Eight semistructured interviews took place with four individuals and four caregivers. Three themes were conceptualized which elucidated key aspects of how digital tools were utilized in disease management: (1) digital tools should lessen the burden of managing a rare disease condition; (2) digital tools should foster community building and promote trust; and (3) digital tools should provide trusted and personalized information to understand the condition and what the future may hold. These results suggest that digital tools play a central role in the lives of individuals with rare disease and their caregivers. Digital tools that centralize trustworthy information, and that bring the relevant community together to interact and promote trust are needed. Genetic counselors can consider these ideal attributes of digital tools when providing resources to individuals and caretakers of rare disease.
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The entire lung epithelium arises from SRY box 9 (SOX9)-expressing progenitors that form the respiratory tree and differentiate into airway and alveolar cells. Despite progress in understanding their initial specification within the embryonic foregut, how these progenitors are subsequently maintained is less clear. Using inducible, progenitor-specific genetic mosaic mouse models, we showed that ß-catenin (CTNNB1) maintains lung progenitors by promoting a hierarchical lung progenitor gene signature, suppressing gastrointestinal (GI) genes, and regulating NK2 homeobox 1 (NKX2.1) and SRY box 2 (SOX2) in a developmental stage-dependent manner. At the early, but not later, stage post-lung specification, CTNNB1 cell-autonomously maintained normal NKX2.1 expression levels and suppressed ectopic SOX2 expression. Genetic epistasis analyses revealed that CTNNB1 is required for fibroblast growth factor (Fgf)/Kirsten rat sarcoma viral oncogene homolog (Kras)-mediated promotion of the progenitors. In silico screening of Eurexpress and translating ribosome affinity purification (TRAP)-RNAseq identified a progenitor gene signature, a subset of which depends on CTNNB1. Wnt signaling also maintained NKX2.1 expression and suppressed GI genes in cultured human lung progenitors derived from embryonic stem cells.
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Linaje de la Célula/genética , Células Madre Embrionarias/metabolismo , Células Epiteliales/citología , Pulmón/embriología , Mucosa Respiratoria/citología , Mucosa Respiratoria/embriología , beta Catenina/fisiología , Animales , Células Cultivadas , Embrión de Mamíferos , Desarrollo Embrionario/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Células Epiteliales/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Embarazo , Mucosa Respiratoria/metabolismo , Transcriptoma , beta Catenina/genéticaRESUMEN
BACKGROUND: Adolescents and young adults in the age range of 13-24 years are at the highest risk of developing HIV infections. As social media platforms are extremely popular among youths, researchers can utilize these platforms to curb the HIV epidemic by investigating the associations between the discourses on HIV infections and the epidemiological data of HIV infections. OBJECTIVE: The goal of this study was to examine how Twitter activity among young men is related to the incidence of HIV infection in the population. METHODS: We used integrated human-computer techniques to characterize the HIV-related tweets by male adolescents and young male adults (age range: 13-24 years). We identified tweets related to HIV risk and prevention by using natural language processing (NLP). Our NLP algorithm identified 89.1% (2243/2517) relevant tweets, which were manually coded by expert coders. We coded 1577 HIV-prevention tweets and 17.5% (940/5372) of general sex-related tweets (including emojis, gifs, and images), and we achieved reliability with intraclass correlation at 0.80 or higher on key constructs. Bivariate and multivariate analyses were performed to identify the spatial patterns in posting HIV-related tweets as well as the relationships between the tweets and local HIV infection rates. RESULTS: We analyzed 2517 tweets that were identified as relevant to HIV risk and prevention tags; these tweets were geolocated in 109 counties throughout the United States. After adjusting for region, HIV prevalence, and social disadvantage index, our findings indicated that every 100-tweet increase in HIV-specific tweets per capita from noninstitutional accounts was associated with a multiplicative effect of 0.97 (95% CI [0.94-1.00]; P=.04) on the incidence of HIV infections in the following year in a given county. CONCLUSIONS: Twitter may serve as a proxy of public behavior related to HIV infections, and the association between the number of HIV-related tweets and HIV infection rates further supports the use of social media for HIV disease prevention.
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Infecciones por VIH/epidemiología , Medios de Comunicación Sociales/normas , Adolescente , Adulto , Femenino , Humanos , Incidencia , Masculino , Reproducibilidad de los Resultados , Estados Unidos , Adulto JovenRESUMEN
We review recent progress in the stem cell biology of the respiratory system, and discuss its scientific and translational ramifications. Several studies have defined novel stem cells in postnatal lung and airways and implicated their roles in tissue homeostasis and repair. In addition, significant advances in the generation of respiratory epithelium from pluripotent stem cells (PSCs) now provide a novel and powerful platform for understanding lung development, modeling pulmonary diseases, and implementing drug screening. Finally, breakthroughs have been made in the generation of decellularized lung matrices that can serve as a scaffold for repopulation with respiratory cells derived from either postnatal or PSCs. These studies are a critical step forward towards the still distant goal of stem cell-based regenerative medicine for diseases of lung and airways.
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Diferenciación Celular , Células Epiteliales/citología , Epitelio/crecimiento & desarrollo , Sistema Respiratorio/citología , Células Madre/citología , Animales , HumanosRESUMEN
Introduction: Mesenteric fibromatosis (intra-abdominal desmoid tumor) is rare, with only a few cases reported in the literature. Clinical symptoms range from asymptomatic, nausea, early satiety, abdominal pain, and gastrointestinal bleeding. Although histologically benign, such a tumor may become locally invasive, and aggressive forms contribute to significant morbidity and mortality. Case Presentation: We report the case of a 52-year-old West African male with a 1-year history of intermittent hematochezia and intermittent bloating. Colonoscopy revealed a 4-mm rectal polyp and internal hemorrhoids. Esophagogastroduodenoscopy revealed a severe duodenal stricture 4-5 cm distal to the ampulla. Further work-up with contrast-enhanced computed tomography of the abdomen and pelvis revealed a 5.0 × 3.7 × 4.3-cm mass within the mesentery, encasing the distal portion of the duodenum. Exploratory laparotomy was performed, and the mass was excised from the jejunum. Histopathology findings and immunohistochemical analysis revealed the diagnosis to be mesenteric fibromatosis (desmoid tumor), positive for nuclear ß-catenin and SMA, and negative expression of STAT6, desmin, caldesmon, pan-cytokeratin, or c-KIT. The Ki67 index is <1%. Conclusion: This case report highlights the diagnostic challenges of mesenteric fibromatosis due to its nonspecific clinical presentation. Recognizing uncommon presentations of mesenteric fibromatosis and risk factors aids in early diagnosis, management, and treatment. Importantly, this also aids in the prevention of complications such as intestinal obstruction, bowel ischemia, and fistula formation.
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Disseminated histoplasmosis is a rare complication of infection due to Histoplasma capsulatum. Typically, histoplasmosis is self-limiting and asymptomatic in infected individuals with immunocompetence. Disseminated disease, however, can arise in high-risk populations with primary or acquired cellular immunodeficiency including HIV/AIDS, transplant recipients, and those undergoing immunosuppressive therapy. Here we describe a unique case of extrapulmonary gastrointestinal histoplasmosis by infiltrative Peyer's patch disease with bone marrow involvement in a transgender HIV-infected woman.
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BACKGROUND: Current risk assessment for ischemic stroke (IS) is limited to clinical variables. We hypothesize that polygenic scores (PGS) of IS (PGSIS) and IS-associated diseases such as atrial fibrillation (AF), venous thromboembolism (VTE), coronary artery disease (CAD), hypertension (HTN), and Type 2 diabetes (T2D) may improve the performance of IS risk assessment. METHODS: Incident IS was followed for 479,476 participants in the UK Biobank who did not have an IS diagnosis prior to the recruitment. Lifestyle variables (obesity, smoking and alcohol) at the time of study recruitment, clinical diagnoses of IS-associated diseases, PGSIS, and five PGSs for IS-associated diseases were tested using the Cox proportional-hazards model. Predictive performance was assessed using the C-statistic and net reclassification index (NRI). RESULTS: During a median average 12.5-year follow-up, 8374 subjects were diagnosed with IS. Known clinical variables (age, gender, clinical diagnoses of IS-associated diseases, obesity, and smoking) and PGSIS were all independently associated with IS (P < 0.001). In addition, PGSIS and each PGS for IS-associated diseases was also independently associated with IS (P < 0.001). Compared to the clinical model, a joint clinical/PGS model improved the C-statistic for predicting IS from 0.71 to 0.73 (P < 0.001) and significantly reclassified IS risk (NRI = 0.017, P < 0.001), and 6.48% of subjects were upgraded from low to high risk. CONCLUSIONS: Adding PGSs of IS and IS-associated diseases to known clinical risk factors statistically improved risk assessment for IS, demonstrating the supplementary value of inherited susceptibility measurement . However, its clinical utility is likely limited due to modest improvements in predictive values.
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Fibrilación Atrial , Diabetes Mellitus Tipo 2 , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/epidemiología , Accidente Cerebrovascular/genética , Diabetes Mellitus Tipo 2/complicaciones , Factores de Riesgo , Medición de Riesgo , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/epidemiología , Fibrilación Atrial/genética , Obesidad/diagnóstico , Obesidad/epidemiología , Obesidad/genéticaRESUMEN
Human small airway epithelial cells (SAECs) immortalized with human telomerase reverse transcriptase were exposed to either a single or multiple doses of α-particles. Irradiated cells showed a dose-dependent cytotoxicity and progressive neoplastic transformation phenotype. These included an increase in saturation density of growth, a greater resistance to N-phosphonoacetyl-L-aspartate, faster anchorage-independent growth, reinforced cell invasion and c-Myc expression. In addition, the transformed cells formed progressively growing tumors upon inoculation into athymic nude mice. Specifically, α-irradiation induced damage to both mitochondrial DNA (mtDNA) and mitochondrial functions in transformed cells as evidenced by increased mtDNA copy number and common deletion, decreased oxidative phosphorylation activity as measured by cytochrome C oxidase (COX) activity and oxygen consumption. There was a linear correlation between mtDNA copy number, common deletion, COX activity and cellular transformation represented by soft agar colony formation and c-Myc expression. These results suggest that mitochondria are associated with neoplastic transformation of SAEC cells induced by α-particles, and that the oncogenesis process may depend not only on the genomes inside the nucleus, but also on the mitochondrial DNA outside the nucleus.
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Partículas alfa , Transformación Celular Neoplásica/efectos de la radiación , Células Epiteliales/efectos de la radiación , Mitocondrias/fisiología , Mitocondrias/efectos de la radiación , Animales , Asparagina/análogos & derivados , Asparagina/farmacología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Cultivadas , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Organofosfonatos/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
The study of chronic brain diseases including Alzheimer's disease in patients is typically limited to brain imaging or psychometric testing. Given the epidemic rise and insufficient knowledge about pathological pathways in sporadic Alzheimer's disease, new tools are required to identify the molecular changes underlying this disease. We hypothesize that levels of specific secreted cellular signaling proteins in cerebrospinal fluid or plasma correlate with pathological changes in the Alzheimer's disease brain and can thus be used to discover signaling pathways altered in the disease. Here we measured 91 proteins of this subset of the cellular communication proteome in plasma or cerebrospinal fluid in patients with Alzheimer's disease and cognitively normal controls to mathematically model disease-specific molecular traits. We found small numbers of signaling proteins that were able to model key pathological markers of Alzheimer's disease, including levels of cerebrospinal fluid ß-amyloid and tau, and classify disease in independent samples. Several of these factors had previously been implicated in Alzheimer's disease supporting the validity of our approach. Our study also points to proteins which were previously unknown to be associated with Alzheimer's disease thereby implicating novel signaling pathways in this disorder.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Proteínas Sanguíneas/análisis , Proteínas del Líquido Cefalorraquídeo/análisis , Proteoma/análisis , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/sangre , Precursor de Proteína beta-Amiloide/líquido cefalorraquídeo , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Comunicación Celular , Femenino , Humanos , Masculino , Modelos Teóricos , Neuroimagen , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Transducción de Señal , Proteínas tau/análisis , Proteínas tau/sangre , Proteínas tau/líquido cefalorraquídeoRESUMEN
Dedicated Neonatal Intensive Care Unit (NICU) follow-up programs are recommended for ongoing surveillance for infants at high-risk for future neurodevelopmental impairment (NDI). Systemic, socioeconomic, and psychosocial barriers remain for referrals and the continued neurodevelopmental follow-up of high-risk infants. Telemedicine can help overcome these barriers. Telemedicine allows standardization of evaluations, increased referral rates, and reduced time to follow-up as well as increased therapy engagement. Telemedicine can expand neurodevelopmental surveillance and support all NICU graduates, facilitating the early identification of NDI. However, with the recent expansion of telemedicine during the COVID-19 pandemic, new barriers related to access and technological support have arisen.
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COVID-19 , Telemedicina , Lactante , Recién Nacido , Humanos , Unidades de Cuidado Intensivo Neonatal , Pandemias , Instituciones AcadémicasRESUMEN
BACKGROUND: The majority of cutaneous squamous cell carcinomas (cSCC) have a favourable prognosis. However, a subset of cases follow an aggressive disease course with progression to metastasis and death. Several histopathological parameters are associated with poor outcomes, but lymphovascular invasion (LVI) has not been well studied. OBJECTIVE: To assess the prognostic significance of LVI in cSCC and determine associations between LVI and cSCC. METHODS: A retrospective review of 486 consecutive cases of cSCC over a 5-year period from a single centre was stratified by the presence or absence of LVI. Logistic regression and multivariate survival analysis were used to determine associations of LVI and prognostic significance of LVI, respectively. FINDINGS: LVI was present in 41 cases (9.2%). LVI was significantly associated with increasing depth of invasion, microanatomical tumour location (subcutis vs. dermis), and tumour dimensions (P < 0.05). Univariate survival analysis revealed significantly lower 2-year overall survival rates for patients with LVI (37.1%) compared with those without (66.6%) (95% CI = 60.6-73.3, P < 0.001). LVI was also found to be an independent marker of poor disease-specific survival (HR = 0.232 (95% CI = 0.090-0.600), P = 0.003), poor overall survival (HR 0.338 (95% CI = 0.184-0.623), P < 0.001) and poor disease-free survival (HR 0.461 (95% CI = 0.230-0.923), P = 0.029) through multivariate analysis. CONCLUSIONS: This study confirms that LVI is an independent poor prognosticator in cSCC, with significantly worse survival indices at 2 years. Future systems of risk stratification for cSCC should incorporate LVI.
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Carcinoma de Células Escamosas , Neoplasias Cutáneas , Humanos , Pronóstico , Carcinoma de Células Escamosas/patología , Estadificación de Neoplasias , Metástasis Linfática , Neoplasias Cutáneas/patología , Estudios Retrospectivos , Invasividad Neoplásica/patologíaRESUMEN
Turner syndrome (TS) is a genetic condition occurring in ~1 in 2,000 females characterized by the complete or partial absence of the second sex chromosome. TS research faces similar challenges to many other pediatric rare disease conditions, with homogenous, single-center, underpowered studies. Secondary data analyses utilizing Electronic Health Record (EHR) have the potential to address these limitations, however, an algorithm to accurately identify TS cases in EHR data is needed. We developed a computable phenotype to identify patients with TS using PEDSnet, a pediatric research network. This computable phenotype was validated through chart review; true positives and negatives and false positives and negatives were used to assess accuracy at both primary and external validation sites. The optimal algorithm consisted of the following criteria: female sex, ≥1 outpatient encounter, and ≥3 encounters with a diagnosis code that maps to TS, yielding average sensitivity 0.97, specificity 0.88, and C-statistic 0.93 across all sites. The accuracy of any estradiol prescriptions yielded an average C-statistic of 0.91 across sites and 0.80 for transdermal and oral formulations separately. PEDSnet and computable phenotyping are powerful tools in providing large, diverse samples to pragmatically study rare pediatric conditions like TS.
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Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism.
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Mitochondrial DNA depleted (ρ(0)) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ(+) HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ(0) cells compared to ρ(+) HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ(+) HSF, but this response was substantially decreased in ρ(0) HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ(+) HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ(0) HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.
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Apoptosis , Cicloheximida/farmacología , ADN Mitocondrial/metabolismo , Rayos gamma , Perfilación de la Expresión Génica , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Biomarcadores/metabolismo , Western Blotting , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Luciferasas/metabolismo , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/efectos de la radiación , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Piel/citología , Piel/efectos de los fármacos , Piel/efectos de la radiaciónRESUMEN
Recent synergistic advances in organ-on-chip and tissue engineering technologies offer opportunities to create in vitro-grown tissue or organ constructs that can faithfully recapitulate their in vivo counterparts. Such in vitro tissue or organ constructs can be utilized in multiple applications, including rapid drug screening, high-fidelity disease modeling, and precision medicine. Here, we report an imaging-guided bioreactor that allows in situ monitoring of the lumen of ex vivo airway tissues during controlled in vitro tissue manipulation and cultivation of isolated rat trachea. Using this platform, we demonstrated partial removal of the rat tracheal epithelium (i.e., de-epithelialization) without disrupting the underlying subepithelial cells and extracellular matrix. Through different tissue evaluation assays, such as immunofluorescent staining, DNA/protein quantification, and electron beam microscopy, we showed that the epithelium of the tracheal lumen can be effectively removed with negligible disruption in the underlying tissue layers, such as cartilage and blood vessel. Notably, using a custom-built micro-optical imaging device integrated with the bioreactor, the trachea lumen was visualized at the cellular level, and removal of the endogenous epithelium and distribution of locally delivered exogenous cells were demonstrated in situ. Moreover, the de-epithelialized trachea supported on the bioreactor allowed attachment and growth of exogenous cells seeded topically on its denuded tissue surface. Collectively, the results suggest that our imaging-enabled rat trachea bioreactor and localized cell replacement method can facilitate creation of bioengineered in vitro airway tissue that can be used in different biomedical applications.
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Ingeniería de Tejidos , Tráquea , Animales , Reactores Biológicos , Cartílago , Ratas , Repitelización , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
Despite the development of metabolism-based therapy for a variety of malignancies, resistance to single-agent treatment is common due to the metabolic plasticity of cancer cells. Improved understanding of how malignant cells rewire metabolic pathways can guide the rational selection of combination therapy to circumvent drug resistance. Here, we show that human T-ALL cells shift their metabolism from oxidative decarboxylation to reductive carboxylation when the TCA cycle is disrupted. The α-ketoglutarate dehydrogenase complex (KGDHC) in the TCA cycle regulates oxidative decarboxylation by converting α-ketoglutarate (α-KG) to succinyl-CoA, while isocitrate dehydrogenase (IDH) 1 and 2 govern reductive carboxylation. Metabolomics flux analysis of T-ALL reveals enhanced reductive carboxylation upon genetic depletion of the E2 subunit of KGDHC, dihydrolipoamide-succinyl transferase (DLST), mimicking pharmacological inhibition of the complex. Mechanistically, KGDHC dysfunction causes increased demethylation of nuclear DNA by α-KG-dependent dioxygenases (e.g., TET demethylases), leading to increased production of both IDH1 and 2. Consequently, dual pharmacologic inhibition of the TCA cycle and TET demethylases demonstrates additive efficacy in reducing the tumor burden in zebrafish xenografts. These findings provide mechanistic insights into how T-ALL develops resistance to drugs targeting the TCA cycle and therapeutic strategies to overcome this resistance.