RESUMEN
Methyl carboxylate esters promote the formation of dimethyl ether (DME) from the dehydration of methanol in H-ZSM-5 zeolite. We employ a multilevel quantum method to explore the possible associative and dissociative mechanisms in the presence, and absence, of six methyl ester promoters. This hybrid method combines density functional theory, with dispersion corrections (DFT-D3), for the full periodic system, with second-order Møller-Plesset perturbation theory (MP2) for small clusters representing the reaction site, and coupled cluster with single, double, and perturbative triple substitution (CCSD(T)) for the reacting molecules. The calculated adsorption enthalpy of methanol, and reaction enthalpies of the dehydration of methanol to DME within H-ZSM-5, agree with experiment to within chemical accuracy (â¼4 kJ mol-1). For the promoters, a reaction pathway via an associative mechanism gives lower overall reaction enthalpies and barriers compared to the reaction with methanol only. Each stage of this mechanism is explored and related to experimental data. We provide evidence that suggests the promoter's adsorption to the Brønsted acid site is the most important factor dictating its efficiency.
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The development of cancer therapies may be improved by the discovery of tumor-specific molecular dependencies. The requisite tools include genetic and chemical perturbations, each with its strengths and limitations. Chemical perturbations can be readily applied to primary cancer samples at large scale, but mechanistic understanding of hits and further pharmaceutical development is often complicated by the fact that a chemical compound has affinities to multiple proteins. To computationally infer specific molecular dependencies of individual cancers from their ex vivo drug sensitivity profiles, we developed a mathematical model that deconvolutes these data using measurements of protein-drug affinity profiles. Through integrating a drug-kinase profiling dataset and several drug response datasets, our method, DepInfeR, correctly identified known protein kinase dependencies, including the EGFR dependence of HER2+ breast cancer cell lines, the FLT3 dependence of acute myeloid leukemia (AML) with FLT3-ITD mutations and the differential dependencies on the B-cell receptor pathway in the two major subtypes of chronic lymphocytic leukemia (CLL). Furthermore, our method uncovered new subgroup-specific dependencies, including a previously unreported dependence of high-risk CLL on Checkpoint kinase 1 (CHEK1). The method also produced a detailed map of the kinase dependencies in a heterogeneous set of 117 CLL samples. The ability to deconvolute polypharmacological phenotypes into underlying causal molecular dependencies should increase the utility of high-throughput drug response assays for functional precision oncology.
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Leucemia Linfocítica Crónica de Células B , Leucemia Mieloide Aguda , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Mutación , Medicina de Precisión , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.
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Neoplasias Óseas , Neoplasias Cerebelosas , Cromotripsis , Osteosarcoma , Animales , Neoplasias Óseas/genética , Línea Celular Tumoral , ADN , Reparación del ADN , Proteínas Hedgehog/genética , Humanos , Ratones , Osteosarcoma/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéuticoRESUMEN
The gene family of protein phosphatases is a rich but under-exploited source of therapeutically validated drug targets modulating signal transduction pathways. Unlike the kinase family, research and development activities have not yet yielded any approved small-molecule drugs against a phosphatase. Approximately 20 years ago, the phosphatase family was classified as undruggable and intractable. This was primarily due to the spectacular failure of the cumulated industry-wide drug discovery efforts to develop PTP1B inhibitors. Recently, allosteric inhibitors against SHP2, a member of the phosphatase family, have entered clinical trails, which has reawakened industry's interest towards this neglected enzyme family. This contribution reviews the recent R&D trends around small-molecule efforts towards phosphatase modulators over the last years, rather than providing an exhaustive review of the field of allosteric phosphatase inhibitors.
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Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.
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Factor 2 de Crecimiento de Fibroblastos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/genética , Interferencia de ARNRESUMEN
Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the ß1- and ß3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of ß-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.
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Factor 2 de Crecimiento de Fibroblastos/metabolismo , Vías Secretoras , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Células HeLa , Humanos , Unión Proteica , Estructura Terciaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Reactivation of fetal hemoglobin expression alleviates the symptoms associated with ß-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.
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In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 µM and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.
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Antiprotozoarios/farmacología , Evaluación Preclínica de Medicamentos , Peroxidasa/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Tiorredoxinas/antagonistas & inhibidores , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/parasitología , Antiprotozoarios/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers.
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Neoplasias , Transactivadores , Animales , Humanos , Ratones , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Transactivadores/antagonistas & inhibidoresRESUMEN
Interactive molecular dynamics simulation in virtual reality (iMD-VR) is emerging as a promising technique in molecular science. Here, we demonstrate its use in a range of fifteen applications in materials science and heterogeneous catalysis. In this work, the iMD-VR package Narupa is used with the MD package, DL_POLY [1]. We show how iMD-VR can be used to: (i) investigate the mechanism of lithium fast ion conduction by directing the formation of defects showing that vacancy transport is favoured over interstitialcy mechanisms, and (ii) guide a molecule through a zeolite pore to explore diffusion within zeolites, examining in detail the motion of methyl n-hexanoate in H-ZSM-5 zeolite and identifying bottlenecks restricting diffusion. iMD-VR allows users to manipulate these systems intuitively, to drive changes in them and observe the resulting changes in structure and dynamics. We make these simulations available, as a resource for both teaching and research. All simulation files, with videos, can be found online (https://doi.org/10.5281/zenodo.8252314) and are provided as open-source material.
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Simulación de Dinámica Molecular , Realidad Virtual , Catálisis , Difusión , Ésteres , LitioRESUMEN
DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.
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The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.
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Proteínas Represoras , Proteína de Retinoblastoma , Ciclo Celular , Humanos , Proteínas Represoras/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/metabolismoRESUMEN
Genetic instability and cellular proliferation have been associated with aurora kinase expression in several cancer entities, including multiple myeloma. Therefore, the expression of aurora-A, -B, and -C was determined by Affymetrix DNA microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive interphase fluorescence in situ hybridization and proliferation of primary myeloma cells by propidium iodine staining. We found aurora-A and -B to be expressed at varying frequencies in primary myeloma cells of different patient cohorts, but aurora-C in testis cell samples only. Myeloma cell samples with detectable versus absent aurora-A expression show a significantly higher proliferation rate, but neither a higher absolute number of chromosomal aberrations (aneuploidy), nor of subclonal aberrations (chromosomal instability). The clinical aurora kinase inhibitor VX680 induced apoptosis in 20 of 20 myeloma cell lines and 5 of 5 primary myeloma cell samples. Presence of aurora-A expression delineates significantly inferior event-free and overall survival in 2 independent cohorts of patients undergoing high-dose chemotherapy, independent from conventional prognostic factors. Using gene expression profiling, aurora kinase inhibitors as a promising therapeutic option in myeloma can be tailoredly given to patients expressing aurora-A, who in turn have an adverse prognosis.
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Apoptosis/efectos de los fármacos , Mieloma Múltiple/patología , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Aurora Quinasa C , Aurora Quinasas , Western Blotting , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Proliferación Celular , Aberraciones Cromosómicas , Terapia Combinada , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Interfase/genética , Mieloma Múltiple/enzimología , Mieloma Múltiple/terapia , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Trasplante de Células Madre , Trasplante Autólogo , Células Tumorales CultivadasRESUMEN
In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.
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Neoplasias Gástricas , Empalme Alternativo , Estudio de Asociación del Genoma Completo , Humanos , Estómago , Proteínas Activadoras de ras GTPasaRESUMEN
OBJECTIVES: Diagnostic tests for SARS-CoV-2 are important for epidemiology, clinical management, and infection control. Limitations of oro-nasopharyngeal real-time PCR sensitivity have been described based on comparisons of single tests with repeated sampling. We assessed SARS-CoV-2 PCR clinical sensitivity using a clinical and radiological reference standard. METHODS: Between March-May 2020, 2060 patients underwent thoracic imaging and SARS-CoV-2 PCR testing. Imaging was independently double- or triple-reported (if discordance) by blinded radiologists according to radiological criteria for COVID-19. We excluded asymptomatic patients and those with alternative diagnoses that could explain imaging findings. Associations with PCR-positivity were assessed with binomial logistic regression. RESULTS: 901 patients had possible/probable imaging features and clinical symptoms of COVID-19 and 429 patients met the clinical and radiological reference case definition. SARS-CoV-2 PCR sensitivity was 68% (95% confidence interval 64-73), was highest 7-8 days after symptom onset (78% (68-88)) and was lower among current smokers (adjusted odds ratio 0.23 (0.12-0.42) p < 0.001). CONCLUSIONS: In patients with clinical and imaging features of COVID-19, PCR test sensitivity was 68%, and was lower among smokers; a finding that could explain observations of lower disease incidence and that warrants further validation. PCR tests should be interpreted considering imaging, symptom duration and smoking status.
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COVID-19 , SARS-CoV-2 , Pruebas Diagnósticas de Rutina , Humanos , Reacción en Cadena de la Polimerasa , ARN Viral , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteolisis , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Daño del ADN , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteómica , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
While clinical environments are highly focused on COVID-19, reports of missed or delayed treatment for conditions that imitate COVID-19, such as pneumonia caused by the fungus Pneumocystis jirovecii, are emerging. Given the uncertain spectrum of COVID-19 presentations and variable sensitivity of laboratory tests for SARS-CoV-2, there is a risk that, without a high index of suspicion, alternative aetiologies may be overlooked while pursuing a diagnosis of COVID-19. The British HIV Association has been calling for the inclusion of HIV testing in all patients admitted to hospital with suspected COVID-19. In this article we reflect on the importance of including HIV testing to prevent avoidable morbidity and mortality in our patients.
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Infecciones Oportunistas Relacionadas con el SIDA , Neumonía por Pneumocystis , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/patología , Infecciones Oportunistas Relacionadas con el SIDA/fisiopatología , Infecciones Oportunistas Relacionadas con el SIDA/terapia , COVID-19 , Infecciones por Coronavirus , Diagnóstico Diferencial , Resultado Fatal , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Persona de Mediana Edad , Pandemias , Pneumocystis carinii , Neumonía por Pneumocystis/diagnóstico , Neumonía por Pneumocystis/patología , Neumonía por Pneumocystis/fisiopatología , Neumonía por Pneumocystis/terapia , Neumonía ViralRESUMEN
N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.
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Retículo Endoplásmico , Regulación de la Expresión Génica , Retículo Endoplásmico/metabolismo , Humanos , Células K562 , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
The aim of this case series is to describe and evaluate our experience of continuous positive airway pressure (CPAP) to treat type 1 respiratory failure in patients with COVID-19. CPAP was delivered in negative pressure rooms in the newly repurposed infectious disease unit. We report a cohort of 24 patients with type 1 respiratory failure and COVID-19 admitted to the Royal Liverpool Hospital between 1 April and 30 April 2020. Overall, our results were positive; we were able to safely administer CPAP outside the walls of a critical care or high dependency unit environment and over half of patients (58%) avoided mechanical ventilation and a total of 19 out of 24 (79%) have survived and been discharged from our care.
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Presión de las Vías Aéreas Positiva Contínua/métodos , Infecciones por Coronavirus , Pandemias , Neumonía Viral , Utilización de Procedimientos y Técnicas/estadística & datos numéricos , Unidades de Cuidados Respiratorios , Insuficiencia Respiratoria , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/terapia , Vías Clínicas/tendencias , Femenino , Humanos , Masculino , Registros Médicos/estadística & datos numéricos , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Consumo de Oxígeno , Neumonía Viral/epidemiología , Neumonía Viral/fisiopatología , Neumonía Viral/terapia , Unidades de Cuidados Respiratorios/métodos , Unidades de Cuidados Respiratorios/organización & administración , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/mortalidad , Insuficiencia Respiratoria/fisiopatología , Insuficiencia Respiratoria/terapia , SARS-CoV-2 , Análisis de Supervivencia , Reino Unido/epidemiologíaRESUMEN
As evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism.