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BACKGROUND: Hyperglycemia is a recognized complication of supraphysiological steroid dosing. There are no consensus guidelines on optimal treatment of steroid-induced hyperglycemia. We assessed the safety of a weight-based insulin protocol for persons treated with supraphysiological doses of steroids to examine the efficacy of using this protocol in patients with diabetes treated with prednisone or methylprednisolone. AREAS OF UNCERTAINTY: There is uncertainty about the optimal dosing of insulin to manage steroid-induced hyperglycemia; thus, a weight-based protocol was created with the goal of reaching euglycemia faster than current practice in persons with diabetes. Variables such as steroid dosing, baseline glycemic control, and duration of steroid use further complicated the ability to manage these patients. INNOVATIONS: The interdisciplinary team of diabetes providers and pharmacists worked together to devise a protocol to manage steroid-induced hyperglycemia with the goal of reducing hyperglycemia while avoiding hypoglycemia, as well as to allow for less reliance on endocrine consultation. The protocol used weight, insulin naivety, renal function, blood glucose measurements, and steroid dosing to determine the insulin dose. There was some evidence to suggest the proportion of blood glucose levels more than 200 mg/dL was lower after protocol initiation compared with before protocol initiation (P = 0.053). Several factors decreased the rate of successful outcomes, including minimal primary team participation, accurate completion of calculations based on the protocol, and initiation of the protocol after several days of hyperglycemia.
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Corticosteroides/efeitos adversos , Peso Corporal/fisiologia , Hiperglicemia/induzido quimicamente , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Glicemia , Protocolos Clínicos , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular , Humanos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Airway hyperresponsiveness (AHR) is a hallmark feature in asthma characterized by exaggerated airway contractile response to stimuli due to increased airway sensitivity and chronic airway remodeling. We have previously shown that allergen-induced AHR in mice is associated with aberrant DNA methylation in the lung genome, suggesting that AHR could be epigenetically regulated, and these changes might predispose the animals to asthma. Previous studies demonstrated that overexpression of phosphodiesterase 4D (PDE4D) is associated with increased AHR. However, epigenetic regulation of this gene in asthmatic airway smooth muscle cells (ASMCs) has not been examined. In this study, we aimed to examine the relationship between epigenetic regulation of PDE4D and ASMC phenotypes. We identified CpG site-specific hypomethylation at PDE4D promoter in human asthmatic ASMCs. We next used methylated oligonucleotides to introduce CpG site-specific methylation at PDE4D promoter and examined its effect on ASMCs. We showed that PDE4D methylation decreased cell proliferation and migration of asthmatic ASMCs. We further elucidated that methylated PDE4D decreased PDE4D expression in asthmatic ASMCs, increased cAMP level, and inhibited the aberrant increase in Ca(2+) level. Moreover, PDE4D methylation reduced the phosphorylation level of downstream effectors of Ca(2+) signaling, including myosin light chain kinase and p38. Taken together, our findings demonstrate that gene-specific epigenetic changes may predispose ASMCs to asthma through alterations in cell phenotypes. Modulation of ASMC phenotypes by methylated PDE4D oligonucleotides can reverse the aberrant ASMC functions to normal phenotypes. This has provided new insight to the development of novel therapeutic options for this debilitative disease.
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Asma/enzimologia , Asma/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Metilação de DNA , Epigênese Genética , Miócitos de Músculo Liso/enzimologia , Sistema Respiratório/enzimologia , Remodelação das Vias Aéreas , Asma/patologia , Cálcio/metabolismo , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Ilhas de CpG , AMP Cíclico/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Miócitos de Músculo Liso/patologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Sistema Respiratório/patologia , Transdução de Sinais , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A novel approach for the determination of parts-per-billion level of 5-hydroxymethyl-2-furaldehyde, furfuryl alcohol, furfural, 2-furyl methyl ketone, and 5-methylfurfural in transformer or rectifier oils has been successfully innovated and implemented. Various extraction methods including solid-phase extraction, liquid-liquid extraction using methanol, acetonitrile, and water were studied. Water was by far the most efficient solvent for use as an extraction medium. Separation of the analytes was conducted using a 4.6 mm × 250 mm × 3.5 µm Agilent Zorbax column while detection and quantitation were conducted with a variable wavelength UV detector. Detection limits of all furans were at 1 ppb v/v with linear ranges range from 5 to 1000 ppb v/v with correlation coefficients of 0.997 or better. A relative standard deviation of at most 2.4% at 1000 ppb v/v and 7.3% at 5 ppb v/v and a recovery from 43% to 90% depending on the analyte monitored were obtained. The method was purposely designed to be environmental friendly with water as an extraction medium. Also, the method uses 80% water and 20% acetonitrile with a mere 0.2 mL/min of acetonitrile in an acetonitrile/water mixture as mobile phase. The analytical technique has been demonstrated to be highly reliable with low cost of ownership, suitable for deployment in quality control labs or in regions where available analytical resources and solvents are difficult to procure.
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Mitochondrial proteins carry out diverse cellular functions including ATP synthesis, ion homeostasis, cell death signaling, and fatty acid metabolism and biogenesis. Compromised mitochondrial quality control is implicated in various human disorders including cardiac diseases. Recently it has emerged that mitochondrial protein turnover can serve as an informative cellular parameter to characterize mitochondrial quality and uncover disease mechanisms. The turnover rate of a mitochondrial protein reflects its homeostasis and dynamics under the quality control systems acting on mitochondria at a particular cell state. This review article summarizes some recent advances and outstanding challenges for measuring the turnover rates of mitochondrial proteins in health and disease. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
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Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteoma , Proteômica , Animais , Humanos , Proteômica/métodosRESUMO
BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.
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Adenosina Difosfato Ribose/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Estrenos/farmacologia , Imidazóis/farmacologia , Íons/química , Íons/metabolismo , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suramina/farmacologia , Canais de Cátion TRPM/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Mitochondrial proteins alter in their composition and quantity drastically through time and space in correspondence to changing energy demands and cellular signaling events. The integrity and permutations of this dynamism are increasingly recognized to impact the functions of the cardiac proteome in health and disease. This article provides an overview on recent advances in defining the spatial and temporal dynamics of mitochondrial proteins in the heart. Proteomics techniques to characterize dynamics on a proteome scale are reviewed and the physiological consequences of altered mitochondrial protein dynamics are discussed. Lastly, we offer our perspectives on the unmet challenges in translating mitochondrial dynamics markers into the clinic.
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Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Miocárdio/metabolismo , Proteoma/metabolismo , Animais , Biomarcadores/metabolismo , Humanos , ProteômicaRESUMO
Mitochondria are a common energy source for organs and organisms; their diverse functions are specialized according to the unique phenotypes of their hosting environment. Perturbation of mitochondrial homeostasis accompanies significant pathological phenotypes. However, the connections between mitochondrial proteome properties and function remain to be experimentally established on a systematic level. This uncertainty impedes the contextualization and translation of proteomic data to the molecular derivations of mitochondrial diseases. We present a collection of mitochondrial features and functions from four model systems, including two cardiac mitochondrial proteomes from distinct genomes (human and mouse), two unique organ mitochondrial proteomes from identical genetic codons (mouse heart and mouse liver), as well as a relevant metazoan out-group (drosophila). The data, composed of mitochondrial protein abundance and their biochemical activities, capture the core functionalities of these mitochondria. This investigation allowed us to redefine the core mitochondrial proteome from organs and organisms, as well as the relevant contributions from genetic information and hosting milieu. Our study has identified significant enrichment of disease-associated genes and their products. Furthermore, correlational analyses suggest that mitochondrial proteome design is primarily driven by cellular environment. Taken together, these results connect proteome feature with mitochondrial function, providing a prospective resource for mitochondrial pathophysiology and developing novel therapeutic targets in medicine.
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Proteínas Mitocondriais/metabolismo , Proteoma , Animais , Cromatografia Líquida , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Espectrometria de Massas em TandemRESUMO
Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing messenger that releases Ca(2+) from endolysosomal organelles. Recent studies showed that NAADP-induced Ca(2+) release is mediated by the two-pore channels (TPCs) TPC1 and TPC2. However, the expression of TPCs and the NAADP-induced local Ca(2+) signals have not been examined in vascular smooth muscle. Here, we found that both TPC1 and TPC2 are expressed in rat pulmonary arterial smooth muscle cells (PASMCs), with TPC1 being the major subtype. Application of membrane-permeant NAADP acetoxymethyl ester to PASMCs elicited a biphasic increase in global [Ca(2+)]i, which was independent of extracellular Ca(2+) and blocked by the NAADP antagonist Ned-19 or the vacuolar H(+)-ATPase inhibitor bafilomycin A1, indicating Ca(2+) release from acidic endolysosomal Ca(2+) stores. The Ca(2+) response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca(2+) signals, including a diffuse increase in cytosolic [Ca(2+)], Ca(2+) sparks, Ca(2+) bursts, and regenerative Ca(2+) release. The diffuse Ca(2+) increase and Ca(2+) bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca(2+) sparks and regenerative Ca(2+) release were inhibited by ryanodine, consistent with cross-activation of loosely coupled ryanodine receptors. Moreover, Ca(2+) release stimulated by endothelin-1 was inhibited by Ned-19, ryanodine, or xestospongin C, suggesting that NAADP-mediated Ca(2+) signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show that NAADP mediates complex global and local Ca(2+) signals. Depending on the physiological stimuli, these diverse Ca(2+) signals may serve to regulate different cellular functions in PASMCs.
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Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Miócitos de Músculo Liso/metabolismo , NADP/análogos & derivados , Artéria Pulmonar/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Carbolinas/farmacologia , Endotelina-1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Masculino , Miócitos de Músculo Liso/citologia , NADP/antagonistas & inibidores , NADP/metabolismo , Oxazóis/farmacologia , Piperazinas/farmacologia , Artéria Pulmonar/citologia , Ratos , Ratos Wistar , Rianodina/farmacologiaRESUMO
Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water ((2)H(2)O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with (2)H(2)O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level (2)H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d(-1) and 0.163 d(-1), respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (≈ 60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.
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Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteólise , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Óxido de Deutério , Meia-Vida , Marcação por Isótopo , CamundongosRESUMO
Rhabdomyolysis is a rare adverse reaction that has a previously established association with levetiracetam use, which selectively binds the synaptic vesicle glycoprotein 2A (SV2A). Its structural analogue, brivaracetam, is a new third-generation antiseizure medication that has a higher affinity for SV2A, and current data suggests it provides a more favorable adverse event profile. Here, however, we report a case of rhabdomyolysis requiring dialysis in which serum creatine kinase level increased rapidly for several days until brivaracetam was discontinued. The delayed creatine kinase peak, rapid decline upon discontinuation of brivaracetam, and prior association of rhabdomyolysis with levetiracetam strongly suggest a causal relationship. To date, there are three reported cases of brivaracetam-associated rhabdomyolysis in the food and drugs administration adverse event reporting system (FAERS). Despite its favorable side effects profile, the use of brivaracetam may be associated with life-threatening rhabdomyolysis.
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Background Andexanet is U.S. Food and Drug Administration (FDA) approved for the reversal of critical bleeding from factor Xa inhibitors and off-label for surgical reversal. Data are lacking on andexanet administration processes. Methods We retrospectively studied patients at a 23-hospital system who received andexanet from November 2019 to March 2023. Abstractors coded demographics, comorbidities, anticoagulant use, andexanet indication, and process times. The primary outcome was presentation-to-andexanet time; diagnosis, ordering, and administration times were calculated. Secondary outcomes included in-hospital postandexanet major thromboembolism/bleeding and mortality. Results In total, 141 patients were analyzed. Andexanet indications were predominantly neurologic bleeding (85.8%). Twenty-four patients (17.0%) were transferred from nontertiary/academic centers to tertiary/academic centers. The median presentation-to-administration time was 192.5 minutes (interquartile range [IQR]: 108.0-337.0 minutes). Components were as follows: 72.5 minutes (IQR: 39.0-137.5 minutes) for bleeding diagnosis; 35.5 minutes (IQR: 0-96.5 minutes) for andexanet ordering; and 53.0 minutes (IQR: 38.5-78.5 minutes) for administration, which was longer at tertiary/academic hospitals (ratio 1.5, 95% confidence interval [CI]: 1.2-2.0, p = 0.002). Gastrointestinal or other critical bleeding (ratio 2.59, 95% CI: 1.67-4.02, p < 0.001), and tertiary/academic center treatment (ratio 1.58, 95% CI: 1.15-2.18, p = 0.005), were associated with increased time. Major thromboembolism, bleeding, and mortality occurred in 10.6, 12.0, and 22.9% of patients, respectively. Conclusions In our cohort, the median presentation-to-administration time was over 3 hours. Cumulative times were longer at tertiary/academic hospitals and for gastrointestinal/other bleeding. Postandexanet major thromboembolism/bleeding occurred more at tertiary/academic hospitals, possibly related to transfers. Prospective studies may elucidate clinical decision-making bottlenecks.
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This work discloses a unique, comprehensive proteomic dataset of Acinetobacter baumannii strains, both resistant and non-resistant to polymyxin B, isolated in Brazil generated using Orbitrap Fusion Lumos. From nearly 4 million tandem mass spectra, the software DiagnoMass produced 240,685 quality-filtered mass spectral clusters, of which PatternLab for proteomics identified 44,553 peptides mapping to 3479 proteins. Crucially, DiagnoMass shortlisted 3550 and 1408 unique mass spectral clusters for the resistant and non-resistant strains, respectively, with only about a third with sequences (and PTMs) identified by PatternLab. Further open-search attempts via FragPipe yielded an additional â¼20% identifications, suggesting the remaining unidentified spectra likely arise from complex combinations of post-translational modifications and amino-acid substitutions. This highlights the untapped potential of the dataset for future discoveries, particularly given the importance of PTMs, which remain elusive to nucleotide sequencing approaches but are crucial for understanding biological mechanisms. Our innovative approach extends beyond the identifications that are typically subjected to the bias of a search engine; we discern which spectral clusters are differential and subject them to increased scrutiny, akin to spectral library matching by comparing captured spectra to themselves. Our analysis reveals adaptations in the resistant strain, including enhanced detoxification, altered protein synthesis, and metabolic adjustments. SIGNIFICANCE: We present comprehensive proteomic profiles of non-resistant and resistant Acinetobacter baumannii from Brazilian Hospitals strains, and highlight the presence of discriminative and yet unidentified mass spectral clusters. Our work emphasizes the importance of exploring this overlooked data, as it could hold the key to understanding the complex dynamics of antibiotic resistance. This approach not only informs antimicrobial stewardship efforts but also paves the way for the development of innovative diagnostic tools. Thus, our findings have profound implications for the field, as far as methods for providing a new perspective on diagnosing antibiotic resistance as well as classifying proteomes in general.
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Acinetobacter baumannii , Polimixinas , Polimixinas/metabolismo , Antibacterianos/farmacologia , Acinetobacter baumannii/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Brasil , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade MicrobianaRESUMO
Physical activity is associated with beneficial adaptations in human and rodent metabolism. We studied over 50 complex traits before and after exercise intervention in middle-aged men and a panel of 100 diverse strains of female mice. Candidate gene analyses in three brain regions, muscle, liver, heart, and adipose tissue of mice indicate genetic drivers of clinically relevant traits, including volitional exercise volume, muscle metabolism, adiposity, and hepatic lipids. Although â¼33% of genes differentially expressed in skeletal muscle following the exercise intervention are similar in mice and humans independent of BMI, responsiveness of adipose tissue to exercise-stimulated weight loss appears controlled by species and underlying genotype. We leveraged genetic diversity to generate prediction models of metabolic trait responsiveness to volitional activity offering a framework for advancing personalized exercise prescription. The human and mouse data are publicly available via a user-friendly Web-based application to enhance data mining and hypothesis development.
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Adaptação Fisiológica , Transcriptoma , Masculino , Pessoa de Meia-Idade , Humanos , Feminino , Camundongos , Animais , Transcriptoma/genética , Obesidade/metabolismo , Aclimatação , Tecido Adiposo/metabolismo , Músculo Esquelético/metabolismoRESUMO
Chronic hypoxia causes pulmonary hypertension with vascular remodeling, increase in vascular tone, and altered reactivity to agonists. These changes involve alterations in multiple Ca(2+) pathways in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that vanilloid (TRPV)- and melastatin-related transient receptor potential (TRPM) channels are expressed in pulmonary arteries (PAs). Here we found that TRPV4 was the only member of the TRPV and TRPM subfamilies upregulated in PAs of chronic hypoxic rats. The increase in TRPV4 expression occurred within 1 day of hypoxia exposure, indicative of an early hypoxic response. TRPV4 in PASMCs were found to be mechanosensitive. Osmo-mechanical stress imposed by hypotonic solution activated Ca(2+) transients; they were inhibited by TRPV4 specific short interfering RNA, the TRPV blocker ruthenium red, and the cytochrome P450 epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide. Consistent with TRPV4 upregulation, the Ca(2+) response induced by the TRPV4 agonist 4α-phorbol 12,13-didecanoate and hypotonicity was potentiated in hypoxic PASMCs. Moreover, a significant myogenic tone, sensitive to ruthenium red, was observed in pressurized endothelium denuded small PAs of hypoxic but not normoxic rats. The elevated basal intracellular Ca(2+) concentration in hypoxic PASMCs was also reduced by ruthenium red. In extension of these results, the development of pulmonary hypertension, right heart hypertrophy, and vascular remodeling was significantly delayed and suppressed in hypoxic trpv4(-/-) mice. These results suggest the novel concept that TRPV4 serves as a signal pathway crucial for the development of hypoxia-induced pulmonary hypertension. Its upregulation may provide a pathogenic feed-forward mechanism that promotes pulmonary hypertension via facilitated Ca(2+) influx, subsequently enhanced myogenic tone and vascular remodeling.
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Hipertensão Pulmonar/fisiopatologia , Soluções Hipotônicas/metabolismo , Hipóxia/fisiopatologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertrofia Ventricular Direita/genética , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Hipóxia/genética , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/metabolismo , Microvasos/fisiopatologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos , Ratos Wistar , Regulação para CimaRESUMO
Myocardial infarction is the leading cause of cardiovascular mortality, with myocardial injury occurring during ischemia and subsequent reperfusion (IR). We previously showed that the inhibition of protein kinase C delta (δPKC) with a pan-inhibitor (δV1-1) mitigates myocardial injury and improves mitochondrial function in animal models of IR, and in humans with acute myocardial infarction, when treated at the time of opening of the occluded blood vessel, at reperfusion. Cardiac troponin I (cTnI), a key sarcomeric protein in cardiomyocyte contraction, is phosphorylated by δPKC during reperfusion. Here, we describe a rationally-designed, selective, high-affinity, eight amino acid peptide that inhibits cTnI's interaction with, and phosphorylation by, δPKC (ψTnI), and prevents tissue injury in a Langendorff model of myocardial infarction, ex vivo. Unexpectedly, we also found that this treatment attenuates IR-induced mitochondrial dysfunction. These data suggest that δPKC phosphorylation of cTnI is critical in IR injury, and that a cTnI/δPKC interaction inhibitor should be considered as a therapeutic target to reduce cardiac injury after myocardial infarction.
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BACKGROUND: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. METHODS: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. RESULTS: Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1ß, TNFα, IL-6). CONCLUSION: These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.
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Dinaminas/fisiologia , Inflamação/etiologia , Macrófagos/fisiologia , Dinâmica Mitocondrial/fisiologia , Proteína Quinase C-delta/fisiologia , Animais , Técnicas de Cultura de Células , Citocinas/metabolismo , Humanos , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologia , Células RAW 264.7RESUMO
Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G(i) and G(q) α-subunit antagonists, respectively, but not by NF449, a selective G(s) α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.
Assuntos
Aorta Torácica/efeitos dos fármacos , Receptores ErbB/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Genisteína/metabolismo , Óxido Nítrico Sintase Tipo III/fisiologia , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/fisiologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Receptores ErbB/genética , Genisteína/farmacologia , Genisteína/uso terapêutico , Hipertensão/enzimologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Técnicas de Cultura de Órgãos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia , Vasodilatação/efeitos dos fármacosRESUMO
Functional neurological disorders (FND) are an important source of healthcare utilization and morbidity. While there are no formal guidelines for treating these disorders, cognitive behavioral therapy (CBT) is emerging as a safe and effective treatment. Currently, there is a global shortage of CBT providers, with only a small subset trained in and comfortable with treating patients with FND. We highlight four types of remote CBT delivery to patients with FND to alleviate the access obstacle: workbooks, internet-guided CBT, app-based CBT, and teletherapy. CBT workbooks and teletherapy have been studied in FND, with preliminary studies suggesting efficacy; internet-guided CBT and app-based CBT have not but have been effectively used in patients with psychiatric disorders, particularly depression, anxiety, and post-traumatic disorders. As these disorders are often comorbid and share overlapping neurobiology with FND, internet-guided CBT and app-based CBT represent promising delivery options of CBT for FND. Although remotely-delivered CBT is unlikely to replace in-person CBT and there are technical and logistical challenges to overcome prior to widespread deployment, it holds promise as an adjunct treatment when in-person CBT is inaccessible. We propose a rational approach to future allocation of remote CBT treatment options and highlight important research gaps to bridge beforehand.
RESUMO
Despite multiple investigational drugs, headache due to subarachnoid hemorrhage (SAH) remains inadequately controlled and requires high opiate utilization. This study investigates the factors associated with increased opiate usage for the management of headache in SAH in the first 14 days of admission, the association between opiate usage and hospital length of stay, and the incidence of opiate consumption during the outpatient follow up. This is a single-center cross-sectional study. A total of 138 patients admitted between January 1, 2017, and May 31, 2019, with a diagnosis of SAH, were identified through a neurocritical care dashboard. Outpatient electronic medical records were evaluated at three months. Statistical analysis included descriptive statistics, Mann-Whitney U test, stepwise regression, and multiple regression analysis. We found that of 138 patients, the majority (90%) were prescribed opiates during their hospitalization, and the mean daily morphine equivalent dosage was 18.74 mg. Steroid usage was associated with an increase in 14-day opiate usage (r = 0.4, p = 0.0001); however, the cerebral spinal fluid profile did not show a statistically significant correlation. Over 14 days, smokers significantly used more opiates compared to nonsmokers (353 mg vs. 184 mg, p = 0.01). In addition, peri-mesencephalic SAH required less morphine compared to aneurysmal SAH (195 mg vs. 283 mg, p = 0.004). Aneurysm clipping was associated with less opiate usage compared to aneurysm coiling (186 vs. 320, p = 0.08). Only the high Hunt and Hess scale score predicted opiate usage, and the high modified Fisher scale score, aneurysmal SAH, and more opiate usage predicted hospital length of stay. A total of 48 patients (42%) suffered from headaches during their outpatient follow-up within three months of discharge; however, only six (5%) were still on opiates. There was a significant association between the amount of opiate used in the first 14 days of admission and the rate of post-discharge headache. In summary, even though patients admitted with SAH require a large amount of opiate for headache management, this did not lead to more opiate consumption in the outpatient setting. However, patients continued to suffer from headaches at three months follow-up. This high opiate consumption is associated with increased hospital length of stay. Studies are needed to identify opiate sparing analgesics that target the pathogenesis of headaches in this patient population.
RESUMO
BACKGROUND: It is widely known that some patients surgically treated for subdural hematoma (SDH) experience neurologic deficits not clearly explained by the acute brain injury or known sequelae like seizures. There is increasing evidence that cortical spreading depolarization (CSD) may be the cause. A recent article demonstrated that CSD occurred at a rate of 15 % and was associated with neurological deterioration in a subset of patients following chronic subdural hematoma evacuation. Furthermore, CSD can lead to ischemia leading to worsening neurologic deficits. CSD is usually detected on electrocorticography (ECoG) and needs cortical strip electrode placement with equipment and expertise that may not be readily available. CASE DESCRIPTION: We report three cases of patients with subdural hematoma (SDH) not undergoing ECoG in whom CSD was suspected to be the cause of their neurologic deficits post evacuation. Extensive workup including neuroimaging and electroencephalography (EEG) were inconclusive. Patients were subsequently treated with ketamine infusion and had resultant neurological recovery. CONCLUSIONS: Ketamine infusion can help reverse neurologic deficits in patients with SDH in whom the deficits are not explained by neuroimaging or electrographic seizure. CSD is a known phenomenon that can result in neurological injury and must remain in the differential diagnosis of such patients. Though only limited cases are discussed (nâ¯=â¯3), this small case series provides the basis for conducting clinical trials evaluating the efficacy of ketamine in improving functional outcome in brain-injured patients demonstrating evidence of CSD.