Papers by carlos alberto bernal barrero
Cell Cycle, Aug 11, 2016
HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral ... more HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of a-ketoglutarate (a-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, a-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.
Revista Argentina de Cardiología, Jul 1, 2015
bioRxiv (Cold Spring Harbor Laboratory), Sep 14, 2022
Tubulin is an essential protein to maintain the cellular structure and for the cell division proc... more Tubulin is an essential protein to maintain the cellular structure and for the cell division process. Inhibiting tubulin polymerization has proven to be an effective method for slowing cancer cell growth. Traditionally, identifying tubulin polymerization inhibitors involved using pure tubulin for in vitro assays or procedures using cells that require cell fixing and anti-tubulin antibody staining. This study explores using a cell line developed via CRISPR genome editing as a cell model to identify tubulin polymerization inhibitors with live cells without using exogenous staining. The cell line has endogenous tagging with fluorescent proteins of β-tubulin and a nuclear protein to facilitate image cellular segmentation by high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine and vincristine, and the presence of phenotypic changes that indicate tubulin polymerization inhibition were confirmed via HCI. A library of 429 kinase inhibitors was screened to discover tubulin polymerization inhibitors and three compounds that inhibit tubulin polymerization were found (ON-01910, HMN-214, and KX2-391). Live cell tracking analysis confirms that depolymerization of tubulin occurs rapidly after compound treatments. These results suggest that CRISPR-edited cells with fluorescent endogenous tagging of b-tubulin can be used to screen larger compound libraries containing diverse chemical families to identify novel tubulin polymerization inhibitors.
Biomolecules, Jan 29, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Free Radical Biology and Medicine, Nov 1, 2016
Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A maj... more Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A major mechanism of chemoresistance involves stabilization of the NRF2 transcription factor. NRF2 levels are normally tightly regulated through interaction with KEAP1, an adaptor that targets NRF2 to the CUL3 E3 ubiquitin ligase for proteolysis. In NSCLC, aberrant NRF2 stabilization is best understood through mutations in NRF2, KEAP1, or CUL3 that disrupt their interaction. Biochemical studies, however, have revealed that NRF2 can also be stabilized through expression of KEAP1-interacting proteins that competitively sequester KEAP1 away from NRF2. Here, we have identified PIDD, as a novel KEAP1interactor in NSCLC that regulates NRF2. We show that this interaction allows PIDD to reduce NRF2 ubiquitination and increase its stability. We also demonstrate that PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo, and that this effect is dependent on NRF2. Finally, we report that NRF2 protein expression in a NSCLC cohort exceeds the typical incidence of combined NRF2, KEAP1, and CUL3 mutations, and that NRF2 expression in this cohort is correlated with PIDD levels. Our data identify PIDD as a new NRF2 regulator, and suggest that variations in PIDD levels contribute to differential chemosensitivities among NSCLC patients. Lung cancer is the leading cause of cancer-related death worldwide, with the majority of patients having non-small cell lung cancer (NSCLC) 1. Fifty-seven percent of lung cancer patients are diagnosed with distant metastasis and have a 5-year survival rate of only 4.7% 2. One of the main obstacles to treatment of NSCLC patients is the common development of resistance to anticancer drugs including platinum-based compounds. Cisplatin [cis-diamminedichloroplatinum(II)] is one of the most widely employed chemotherapies in oncology. Cisplatin and platinum-based analogs are currently used to treat many malignancies, including lung, ovarian, head and neck, bladder, and testicular cancer 3. However, toxicity and resistance have become major barriers limiting the use and efficacy of platinum-based agents. Multiple mechanisms have been shown to be involved in the protection of cancer cells against anticancer drugs, including phase II detoxifying enzyme genes, antioxidant genes, and drug efflux protein genes 4-7. NF-E2-related factor 2 (NRF2) is a transcription factor whose activation in cancer cells has been implicated in resistance to chemotherapy 8. It directly regulates a battery of downstream anti-oxidant genes, including (1) intracellular redox-balancing proteins (glutamate cysteine ligase, GCL; heme oxygenase-1, HO-1), (2) xenobiotic metabolizing enzymes (NAD[P]H quinone oxidoreductase-1, NQO1), and (3) transporters (multidrug resistance-associated proteins, MRPs). Collectively, these genes function in a vast array of processes to protect
The CRISPR journal, Nov 30, 2021
The lack of efficient tools to label multiple endogenous targets in cell lines without staining o... more The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.
Cyclin-dependent kinase 9 (CDK9) belongs to the class of CDKs involved in transcription regulatio... more Cyclin-dependent kinase 9 (CDK9) belongs to the class of CDKs involved in transcription regulation along with CDK7, 8, 10-13. Earlier works have established that CDK9 is the catalytic subunit of P-TEFb, and a transcriptional activator. CDK9 inhibition holds promise for patients belonging to Chronic Lymphocytic Leukemia (CLL) and BRD4-NUT-rearranged NUT midline carcinoma (NMC) groups. It is therefore fundamentally important to understand CDK9 mediated gene regulation. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is known to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors. Additionally, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. It is however not known whether CDK9 has any transcriptional repression activity. Earlier work from our lab demonstrated that long-term CDK9 inhibition leads to the activation of BRG1, an ATP-dependent nucleosome-remodeling complex from the SWI/SNF family of proteins. We therefore hypothesized that CDK9 directly phosphorylates BRG1 and regulates its activity. Using Co-immunoprecipitation (Co-IP) assay we discovered that CDK9 associates with BRG1 endogenously in colon cancer cells. We tested the association of these proteins more rigorously by transiently over-expressing either FLAG-tagged or GFP-tagged CDK9 in HEK293T cells and performed immunoprecipitation using BRG1 antibody. CDK9 was found to co-precipitate with BRG1. We validated this via reciprocal Co-IPs. We performed an in vitro kinase assay using purified CDK9 and BRG1 proteins to demonstrate that CDK9 directly phosphorylates BRG1 and that inhibition of CDK9 leads to reduced phosphorylation of BRG1. We validated these results in vivo in colon cancer cells. Furthermore, our LC-MS/MS data lead to the identification of novel phosphorylation sites in BRG1 following CDK9 inhibition. These findings provide a potential mechanism for the transcriptional repressor activity associated with CDK9 and may be relevant for developing therapies targeting this transcriptional regulator. Citation Format: Somnath Pandey, Hanghang Zhang, Carlos A. Barrero, Salim Merali, Xavier Graña, Jean-Pierre Issa. CDK9 phosphorylates BRG1 chromatin remodeler [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2995.
Argentine Journal of Cardiology, Sep 29, 2011
Revista Argentina de Cardiología, Jul 1, 2015
Revista Argentina de Cardiología, 2015
Argentine Journal of Cardiology, Nov 11, 2011
Este proyecto estudia la exploracion de las proteinas y el papel de los aminoacidos a la respuest... more Este proyecto estudia la exploracion de las proteinas y el papel de los aminoacidos a la respuesta inmunologica de las moleculas en los procesos invasivos. La base experimental en este proyecto, es la modificacion de las propiedades quimicas del enlace peptidico.
Medicine and Science in Sports and Exercise, May 1, 2017
respectively, p<0.05). Maximal MSST performance during week 1 of BMT was strongly correlated with... more respectively, p<0.05). Maximal MSST performance during week 1 of BMT was strongly correlated with load carriage performance at both week 1 and 11 of BMT (r 2 =-0.676 and-0.520 respectively, p<0.05). CONCLUSIONS: The results showed that performance in the MSST and push-ups were moderately to strongly correlated with load carriage performance. The predictive utility of these generic fitness tests decreased over BMT. There was a weak correlation between push-up performance and occupationally-relevant muscular strength performance. Both manual handling and load carriage are enduring requirements for Army personnel. In fact, a recent review of physically demanding tasks across all Army employment categories revealed that muscular strength was the dominant physical capacity. These results indicate that the current Australian Army recruit physical barrier tests do not predict the ability of male candidates to perform key occupational tasks (i.e. manual handling). It is therefore recommended that an additional test is incorporated into the recruit barrier test battery that assesses and/or predicts whole-body muscular strength performance.
International Journal of Exercise Science: Conference Proceedings, 2017
International Journal of Molecular Sciences
EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue si... more EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein–protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as “DTX3c”), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the E...
Revista Argentina de Cardiología, 2018
Introducción: La prevención secundaria en pacientes menores de 76 años que han padecido un evento... more Introducción: La prevención secundaria en pacientes menores de 76 años que han padecido un evento vascular o han sido revascularizados incluye el uso de estatinas en altas dosis. Objetivo: Evaluar la adherencia al año a dicho tratamiento instituido desde el alta de la internación en UCO. Materiales y métodos: Estudio prospectivo de pacientes consecutivos durante el período enero-noviembre de 2015. Seguimiento (mediana) 9 meses. Resultados: Doscientos diez pacientes. El 83% eran hombres. La edad (mediana) alcanzó los 59 años (52-67,5). El 74,5% tuvo alta con atorvastatina a 40 mg/día; un 19%, con rosuvastatina a 20 mg/día; un 2,7%, con atorvastatina a 80 mg/día; y un 3,9%, con rosuvastatina a 40 mg/día. Un 50% de los pacientes continuaron tomando estatinas de alta intensidad, 28% redujeron la dosis y 22% abandonaron el tratamiento. Conclusiones: Solo la mitad de los pacientes con alto riesgo vascular o procedimiento de revascularización reciente mantiene el tratamiento al año.
Pharmaceutics, 2021
Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regul... more Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Posiphen’s safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protei...
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Papers by carlos alberto bernal barrero