Mitochondrial free [Ca2+] levels and the permeability transition

Producción CientíficaMitochondrial Ca2+ activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca2+] ([Ca2+]M) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca2+]M from increasing above 2–3 M. Instead, using low-Ca2+-affinity aequorin probes, we have measured [Ca2+]M values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca2+]M to levels of 0.5–1mM was actually observed at any phosphate concentration (0–10mM) during continuous perfusion of 3.5–100 MCa2+-buffers. In spite of this high and maintained (>10 min) [Ca2+]M, mitochondria retained functionality and the [Ca2+]M drop induced by a protonophore was fully reversible. In addition, this high [Ca2+]M did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca2+]M. In conclusion [Ca2+]M levels of 0.5–1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators

The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations

Producción CientíficaThere is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in nonexcitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations

Calcium dynamics in catecholamine-containing secretory vesicles

Producción CientíficaWe have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca2+] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca2+] around 40 M. Cell stimulation with either ATP, caffeine or high-K+ depolarization increased cytosolic [Ca2+] and decreased secretory granule [Ca2+] ([Ca2+]SG). Inositol-(1,4,5)- trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca2+ from the granules. Changes in cytosolic [Na+] (0–140 mM) or [Ca2+] (0–10 M) did not modify either ([Ca2+]SG). Instead, [Ca2+]SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca2+]SG, while cytosolic alcalinization reversibly increased [Ca2+]SG. These results are consistent with the operation of a H+/Ca2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K+ on [Ca2+]SG, because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca2+]SG was reversible in 10–15 min even in the absence of cytosolic Ca2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca2+ buffer. This large store buffers [Ca2+]SG changes in the long-term but allows highly dynamic free [Ca2+]SG changes to occur in seconds or minutes

Caracterización de pacientes sicklémicos en el Hospital Pediátrico “Hermanos Cordové”, papel de la hidroxiurea

Introduction: Sickle cell disease is a hematological and hereditary disease whose generic name was assigned to a group of genetic alterations characterized by the predominance of hemoglobin S. Objective: To describe clinically sicklemic patients and hydroxyurea therapy. Material and Method: An observational, descriptive, retrospective study on the morbidity of sicklemic patients graduated from the Oncohematology ward of the Hermanos Cordové Provincial Pediatric Teaching Hospital during the period from February 2013 to February 2018 was carried out. The population consisted of 37 sicklemic patients. Results: It was found that the predominant age was from 10 to 18 years with a total of 26 patients for 70.2%, with a predominance of males representing 67.6%. The Bartolomé Masó municipality was the one that contributed the most cases with 12 patients, there was a predominance of mixed-race patients represented by 24 patients for 64.9%. Painful vaso-occlusive crises predominated with 76 cases, and seizure crises with seizures with 12 patients for 100%. Conclusions: High doses of HU are not necessary to improve the clinical picture of sickle cell anemia, when administering low doses of the drug there is less risk that the patient presents toxic manifestations secondary to the drug. Introducción: la drepanocitosis es un evento morboso hematológico y hereditario cuyo nombre genérico fue designado a un grupo de alteraciones genéticas caracterizadas por predominio de la hemoglobina S. Objetivo: describir clínicamente a los pacientes sicklémicos y la terapéutica con hidroxiurea. Material y Método: se realizó un estudio observacional, descriptivo retrospectivo sobre la morbilidad de los pacientes sicklémicos egresados de la sala de Oncohematología del Hospital Provincial Pediátrico Docente Hermanos Cordové durante el periodo de febrero 2013 a febrero 2018. La población quedo constituida por 37 pacientes sicklémicos. Resultados: se encontró que la edad predominante fue de 10 a 18 años con un total de 26 pacientes para un 70,2 %, con un predominio del sexo masculino (67,6 %). El municipio Bartolomé Masó fue el que más caso aportó con 12 pacientes, hubo un predominio de los pacientes de raza mestiza representado por 24 pacientes (64,9 %). Predominaron las crisis vasooclusivas dolorosas con 76 casos, y de las crisis hematológicas las de secuestro con 12 pacientes (100 %).  Conclusiones: no son necesarias dosis elevadas de hidroxiurea para mejorar el cuadro clínico de la anemia drepanocítica, al administrar dosis bajas del medicamento existe menos riesgo de que el paciente presente manifestaciones tóxicas secundarias a la droga

Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules

Producción CientíficaThe secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50–100 lm Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular [Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+ during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.2015-09-1

Usefulness of Serial Multiorgan Point-of-Care Ultrasound in Acute Heart Failure: Results from a Prospective Observational Cohort.

Background and Objectives: Acute heart failure (AHF) is a common disease and a cause of high morbidity and mortality, constituting a major health problem. The main purpose of this study was to determine the impact of multiorgan ultrasound in identifying pulmonary hypertension (PH), a major prognostic factor in patients admitted due to AHF, and assess whether there are significant changes in the venous excess ultrasonography (VE US) score or femoral vein Doppler at discharge. Materials and Methods: Patients were evaluated with a standard protocol of lung ultrasound, echocardiography, inferior vena cava (IVC) and hepatic, portal, intra-renal and femoral vein Doppler flow patterns at admission and on the day of discharge. Results: Thirty patients were enrolled during November 2021. The mean age was seventy-nine years (Standard Deviation–SD 13.4). Seven patients (23.3%) had a worsening renal function during hospitalization. Regarding ultrasound findings, VE US score was calculated at admission and at discharge, unexpectedly remaining unchanged or even worsened (21 patients, 70.0%). The area under the curve for the lung score was 83.9% (p = 0.008), obtaining a cutoff value of 10 that showed a sensitivity of 82.6% and a specificity of 71.4% in the identification of intermediate and high PH. It was possible to monitor significant changes between both exams on the lung score (16.5 vs. 9.3; p < 0.001), improvement in the hepatic vein Doppler pattern (2.4 vs. 2.1; p = 0.002), improvement in portal vein Doppler pattern (1.7 vs. 1.4; p = 0.023), without significant changes in the intra-renal vein Doppler pattern (1.70 vs. 1.57; p = 0.293), VE US score (1.3 vs. 1.1; p = 0.501), femoral vein Doppler pattern (2.4 vs. 2.1; p = 0.161) and IVC collapsibility (2.0 vs. 2.1; p = 0.420). Conclusions: Our study results suggest that performing serial multiorgan Point-of-Care ultrasound can help us to better identify high and intermediate probability of PH patients with AHF. Currently proposed multi-organ, venous Doppler scanning protocols, such as the VE US score, should be further studied before expanding its use in AHF patients.post-print2977 K

The Impact of Different Lung Ultrasound Protocols in the Assessment of Lung Lesions in COVID-19 Patients: Is There an Ideal Lung Ultrasound Protocol?.

Background In the past months, several lung ultrasonography (LUS) protocols have been proposed, mainly on previously validated schemes independent of coronavirus disease 2019 (COVID-19). Objectives The main purpose of this study was to determine the impact and accuracy of different LUS protocols proposed in COVID-19. Methods Patients were evaluated with a standard sequence of LUS scans in 72 intercostal spaces along 14 anatomic lines in the chest. A scoring system of LUS findings was reported and then analyzed separately according to each proposed LUS protocol zones. This score was then correlated to a validated Pulmonary Inflammation Index (PII) on chest Computed Tomography (CT). Results Thirty-two patients were enrolled. The most frequent pattern was ground-glass opacities in the chest X-ray (53.1%), chest CT (59.1%) and subpleural or lobar consolidations (40.8%) in the posteroinferior areas (p < 0.001) on LUS. The Interclass Correlation Coefficient (ICC) was significantly correlated with almost every protocol analyzed except the 8-zone (p = 0.119) and the 10-zone protocol that only included one posterior point (p = 0.052). The highest ICC was obtained with a 12-zone protocol (ICC 0.500; p = 0.027) and decreased as more points were included. Conclusions In conclusion, our study results suggest that performing an ultrasound protocol with 12-zone scanning, including the superior and inferior areas of the anterior, lateral and posterior regions of the chest was consistent with higher ICC and higher degree of concordance with CT. We emphasize the need of a more standardization technique to further implement and develop this imaging modality in COVID-19post-print1035 K

Genomics And Susceptibility Profiles Of Extensively Drug-resistant Pseudomonas Aeruginosa Isolates From Spain

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (> 95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the beta-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in beta-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance