ACV isquémico en población joven: ¿existe un enfoque diferencial del riesgo? / Ischemic stroke in a young population: is there a differential approach to risk factors?

Introducción: El ataque cerebrovascular isquémico en el adulto joven se define como aquel que ocurre en la población entre los 18 y los 55 años, y representa el 15-18 % de todos los ACV isquémicos. Los factores de riesgo en jóvenes son comunes a los encontrados en adultos mayores. El objetivo de este trabajo es describir las características clínicas y los factores de riesgo de una población menor de 55 años con ACV isquémico atendida en un centro de referencia hospitalario en Colombia. Materiales y métodos: Estudio descriptivo, retrospectivo de corte transversal en pacientes entre los 18 y los 55 años; se incluyeron 100 pacientes sobrevivientes a un primer ACV isquémico agudo confirmado por neuroimagen, atendidos entre enero de del 2019 y noviembre del 2021. Resultados: De 1023 pacientes con diagnóstico de ACV isquémico agudo, el 9,8 % fueron adultos jóvenes. La media de edad fue de 45 ± 8,7 años, y el 59 % de estos pacientes fueron hombres. Discusión: Los factores de riesgo "tradicionales" se presentan en la mayoría de los jóvenes con ACV isquémico. La hipertensión arterial se mantiene como el antecedente más frecuente. Las mujeres presentan eventos de mayor severidad y peor desenlace funcional. Conclusión: Los pacientes mayores de 45 años tienen un perfil de factores de riesgo similar a lo observado en adultos mayores con ACV, mientras que en los menores de 45 años se encuentra un perfil de factores de riesgo diferente que plantea un diagnóstico etiológico particular de esta población.

Introduction: Ischemic stroke in young adults is defined as occurring in individuals between the ages of 18 and 55, representing 15-18 % of all ischemic strokes. Risk factors in young adults are similar to those found in older adults. The objective of this study is to describe the clinical characteristics and risk factors of a population under 55 years of age with ischemic stroke treated at a hospital reference center in Colombia. Materials and methods: Descriptive, retrospective cross-sectional study in patients between 18 and 55 years old. A total of 100 patients between 18 and 55 years old who survived a first confirmed acute ischemic stroke, as confirmed by neuroimaging, were included. The study period was from January 2019 to November 2021. Results: Out of 1023 patients diagnosed with acute ischemic stroke, 9.8 % occurred in young adults. The mean age was 45 ± 8.7 years, of which 59 % were male. Discussion: "Traditional" risk factors are present in the majority of young adults with ischemic stroke. Hypertension remains the most common antecedent. Women experience more severe events and worse functional outcomes. Conclusion: Patients over 45 years old have a risk factor profile similar to what is observed in older adults with stroke, while in those under 45, a different risk factor profile is found, which poses a particular etio-logical diagnosis for this population.

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1).

Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure-function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate-gating relationship in TMEM16A remain less explored. Here, we study the gating-permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN- mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN-. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine.

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism.

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

In vitro and in silico characterization of the inhibition of Kir4.1 channels by aminoglycoside antibiotics.

BACKGROUND AND PURPOSE: Aminoglycoside antibiotics are positively charged molecules that are known to inhibit several ion channels. In this study, we have shown that aminoglycosides also inhibit the activity of Kir4.1 channels. Aminoglycosides inhibit Kir4.1 channels by a pore-blocking mechanism, plugging the central vestibule of the channel. EXPERIMENTAL APPROACH: Patch-clamp recordings were made in HEK-293 cells transiently expressing Kir4.1 channels to analyse the effects of gentamicin, neomycin and kanamycin. In silico modelling followed by mutagenesis were realized to identify the residues critical for aminoglycosides binding to Kir4.1. KEY RESULTS: Aminoglycoside antibiotics block Kir4.1 channels in a concentration- and voltage-dependent manner, getting access to the protein from the intracellular side of the plasma membrane. Aminoglycosides block Ki4.1 with a rank order of potency as follows: gentamicin ˃ neomycin ˃ kanamycin. The residues T128 and principally E158, facing the central cavity of Kir4.1, are important structural determinants for aminoglycosides binding to the channel, as determined by our in silico modelling and confirmed by mutagenesis experiments. CONCLUSION AND IMPLICATIONS: Kir4.1 channels are also target of aminoglycoside antibiotics, which could affect potassium transport in several tissues.

Regulation of the Ca2+-activated chloride channel Anoctamin-1 (TMEM16A) by Ca2+-induced interaction with FKBP12 and calcineurin.

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.

Voltage-Dependent Protonation of the Calcium Pocket Enable Activation of the Calcium-Activated Chloride Channel Anoctamin-1 (TMEM16A).

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.

The voltage-sensitive cardiac M2 muscarinic receptor modulates the inward rectification of the G protein-coupled, ACh-gated K+ current.

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Regulation of Kv2.1 channel inactivation by phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.

Phytochemicals genistein and capsaicin modulate Kv2.1 channel gating.

BACKGROUND: Phytochemicals are a large group of plant-derived compounds that have a broad range of pharmacological effects. Some of these effects are derived from their action on transport proteins, including ion channels. The present study investigates the effects of the phytochemicals genistein and capsaicin on voltage-gated potassium Kv2.1 channels. METHODS: The whole-cell patch clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by genistein and capsaicin. RESULTS: Both phytochemicals had a profound effect on the gating properties of Kv2.1 channels; the voltage dependence of activation and inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was accelerated, and the recovery from inactivation was delayed. Moreover, genistein and capsaicin inhibited Kv2.1 currents in a concentration dependent manner. CONCLUSION: This study effectively demonstrated the inhibitory effects of genistein and capsaicin on Kv2.1 channels. As Kv2.1 channels play a prominent role in glucose-stimulated insulin secretion, our findings contribute to our understanding of the putative mechanism by which these phytochemicals exert their reported hypoglycemic effects.

High-potency block of Kir4.1 channels by pentamidine: Molecular basis.

Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.

Inhibition of Kir4.1 potassium channels by quinacrine.

Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3µM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.

Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism.

Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.

Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

KEY POINTS: The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. ABSTRACT: Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5  m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0  m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5  m, but 10-9.0  m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .

Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes.

BACKGROUND: The rapid delayed rectifier K+ current (IKr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. METHODS: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. RESULTS: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. CONCLUSIONS: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Neumotórax espontáneo en embarazada: Reporte de un caso tratado por videotoracoscopia y revisión de la literatura / Spontaneous pneumothorax during pregnancy: case report of a patient treated with videothoracoscopy and literature review

RESUMEN El neumotórax espontáneo primario es la presencia de aire en la cavidad pleural como consecuencia de la ruptura de bulas o blebs subpleurales en un pulmón que por otro lado está sano y sin antecedentes traumáticos. Es más frecuente en hombres que en mujeres y es raro en el embarazo, habiéndose publicado menos de 60 casos en la literatura. El objetivo es reportar un neumotórax espontáneo en una embarazada y realizar una revisión del tema. El caso corresponde a una mujer de 19 años, primigestante que presenta un neumotórax espontáneo primario tratado inicialmente con pleurotomía, el cual evoluciona satisfactoriamente, pero a las 24 horas ser retirada esta, presenta recidiva por lo que se realiza videotoracoscopia con resección de bulas y pleurodesis. Evoluciona adecuadamente, se da de alta en buenas condiciones y posteriormente lleva a cabo su trabajo de parto vaginal sin complicaciones. El tratamiento del neumotórax en el embarazo es igual al de los pacientes no obstétricos. Los neumotórax espontáneos recurrentes, los persistentes, los con fuga aérea por el tubo más allá del cuarto día y los bilaterales son indicaciones de procedimiento quirúrgico por toracotomía o videotoracoscopia. Se debe considerar el diagnóstico en cualquier embarazada con dolor torácico agudo, disnea súbita o antecedentes de neumotórax previo y este debe ser confirmado con radiografía de tórax con la adecuada protección del feto. Su reconocimiento y manejo es esencial para evitar complicaciones a la madre y al feto. El tratamiento quirúrgico por videotoracoscopia fue seguro en este caso. MÉD.UIS. 2016;29(3):101-5.

ABSTRACT Primary spontaneous pneumothorax is the presence of air in the pleural cavity as a consequence of a rupture of bullae or subpleural blebs in an otherwise healthy lung, without a clear history of trauma. Is more frequent in men than in women, and rarely presents during pregnancy, less than 60 cases has been reported in literature. The objective is to report the case of a 19-year-old primiparous woman who presents spontaneous pneumothorax treated initially with pleurostomy. Initial evolution is satisfactory, but 24 hours after withdrawal of chest tube, patient recurs. Patient is managed with videothoracoscopic bullectomy followed by pleurodesis. The procedure was well tolerated and is discharged in optimum condition and subsequently goes into labor, giving birth without any complications. In conclusion, treatment of spontaneous pneumothorax during pregnancy can be safely managed in the same way as non-obstetric patients. Recurring, persistent, or with air leak beyond 4 days and bilateral spontaneous pneumothorax, are indication for thoracic surgery. The diagnosis of spontaneous pneumothorax must be considered in any pregnant woman with acute thoracic pain, sudden onset dyspnoea and past medical history of it. The diagnosis must be confirmed with chest X ray, considering fetus protecting measures. Recognition and opportune treatment of spontaneous pneumothorax in the pregnant woman is essential to avoid maternal or fetal complications. Videothoracoscopic treatment has been proven safe in this case. MÉD.UIS. 2016;29(3):101-5.

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 µM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

The molecular basis of chloroethylclonidine block of inward rectifier (Kir2.1 and Kir4.1) K(+) channels.

BACKGROUND: Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Kir channels dysfunction cause several diseases in brain, ear, heart, muscle, kidney and pancreas, and developmental abnormalities. Therefore, a better understanding of Kir channels pharmacology is desirable. In this study we characterized the electrophysiological and molecular basis of the inhibition produced by the α-adrenergic agonist/antagonist chloroethylclonidine of the currents generated by wild type and mutant Kir2.1 and Kir4.1 channels heterologously expressed in HEK293 cells. METHODS: Macroscopic currents were recorded using the patch clamp technique in the inside out configuration. RESULTS: We found that chloroethylclonidine inhibits the Kir2.1 and Kir4.1 channels in a voltage-dependent manner by interacting with pore facing residues in the cytoplasmic and transmembrane domains, respectively. Site-directed mutagenesis experiments demonstrate that chloroethylclonidine interact with Kir2.1 channels in the cytoplasmic pore involving the E224, E299, D255 and D259 residues, whereas in Kir4.1channels T128 and E158 residues located in the transmembrane pore are important for the chloroethylclonidine effect. CONCLUSIONS: Overall, our results suggest that differences in the cavity of Kir channels are determinants in its interactions with chloroethylclonidine.

Modulation of the voltage-gated potassium channel Kv2.1 by the anti-tumor alkylphospholipid perifosine.

BACKGROUND: The aim of the present study was to assess the effects of perifosine-a third generation alkylphospholipid analog with anti-tumor properties-on the activity of Kv2.1 channels. METHODS: The whole-cell patch clamp technique was applied to follow the modulatory effect of perifosine on Kv2.1 channels expressed in HEK293 cells. RESULTS: Obtained data provide evidence that perifosine application decreases the whole cell Kv2.1 currents in a concentration-independent manner. Perifosine induces a hyperpolarizing shift in the voltage dependence of Kv2.1 channels inactivation without altering the voltage dependence of channels activation. The kinetics of Kv2.1 closed-state inactivation was accelerated by perifosine, with no significant effects on the recovery rate from inactivation. CONCLUSIONS: Taken together, these results show that perifosine modified the Kv2.1 inactivation gating resulting in a decrease of the current amplitude. These data will help to elucidate the mechanism of action of this promising anti-cancer drug on ion channels and their possible implications.

Autoverification of the automated blood cell counter (CBC) in a reference laboratory in Bogota, Colombia / Autoverificação do contador de células automatizado de sangue (CBC) em um laboratório de referência em Bogotá, Colômbia

ABSTRACT Introduction: The clinical laboratory is part of the group of actors in health systems that are under increasing pressure by users and administrators to increase their productivity in order to respond efficiently to the increased volume of patients, optimizing costs and professional time. This pressure forced laboratories to perform a full review of their procedures and develop technical, logistical and computational tools to enable excellent response times. Objective: This study aimed to evaluate the implementation of the automated blood cell counter autoverification process and its impact on the safety of patients. Methods: Verification rules were designed in the connectivity software, based on manual validation criteria for laboratory professionals, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) Guideline Auto10-A and the International Consensus Group for Hematology Review (ISLH). The autoverification percentage was established, and non-conforming product (NCP) percentages were estimated before and after the procedure. Pilot tests were also performed in different days so as to adjust the process. Results: 53.4% of automated blood cell counters autoverification were achieved, and, subsequently in the audit of 18 months, 60% was reached due to verification adjustments in the delta programmed filter. The NCPs rose from 0.065% to 0.0036% from the beginning to the end of the process. Conclusion: The autoverification process enabled to reduce the variability associated with human intervention, therefore the professional is able to focus on the pathological report analysis, reducing the risk of errors and advocating greater importance on patient safety.

RESUMO Introdução: O laboratório clínico é parte do grupo de atores nos sistemas de saúde, que são cada vez mais pressionados por usuários e administradores para aumentar sua produtividade a fim de responder de forma eficiente ao aumento do volume de pacientes, otimizando custos e tempo dos funcionários. Essa pressão forçou laboratórios para efetuar uma revisão completa de seus procedimentos, bem como desenvolver instrumentos técnicos, logísticos e computacionais para permitir excelentes tempos de resposta. Objetivo: Neste estudo, a implementação do processo automatizado de autoverificação do hemograma e seu impacto sobre a segurança do paciente são avaliados. Métodos: Regras de verificação foram construídas no software de conectividade com base em critérios de validação manual dos profissionais de laboratório, de acordo com as orientações do Clinical and Laboratory Standards Institute (CLSI) de Guideline Auto10-A e do International Consensus Group for Hematology Review (ISLH). A percentagem de autoensaio foi estabelecida e as de produtos não conformes (PNC), estimadas antes e depois do procedimento. Testes piloto foram realizados em dias diferentes para ajustar o processo. Resultados: Cinquenta e três por cento da autoverificação dos hemogramas automatizados foram alcançados e, posteriormente, na auditoria dos 18 meses, foram obtidos 60% devido a ajustes na verificação do filtro delta programado. Os PNCs aumentaram de 0,065% a 0,0036% desde o início até ao final do processo. Conclusão: O processo do autoverificação ajudou a reduzir a variabilidade associada à intervenção humana, portanto o profissional pode concentrar-se na análise de relatórios patológicos, reduzindo o risco de erros e dando maior importância para a segurança do paciente.