Distinct pathophysiological characteristics in developing muscle from patients susceptible to malignant hyperthermia.

BACKGROUND: Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine-halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility. METHODS: Patients with malignant hyperthermia susceptibility were classified into subgroups HH and HS (positive to halothane only and positive to both caffeine and halothane). The effects on [Ca2+]cyto of halothane concentrations between 0.5 and 3 % were measured in myotubes and compared with CHCT responses of muscle. A clinical index that summarises patient symptoms was determined for 67 patients, together with a calcium index summarising resting [Ca2+]cyto and spontaneous and electrically evoked Ca2+ events in their primary myotubes. RESULTS: Halothane-hypersensitive myotubes showed a higher response to halothane 0.5% than the caffeine-halothane hypersensitive myotubes (P<0.001), but a lower response to higher concentrations, comparable with that used in the CHCT (P=0.055). The HH group had a higher calcium index (P<0.001), but their clinical index was not significantly elevated vs the HS. Principal component analysis identified electrically evoked Ca2+ spikes and resting [Ca2+]cyto as the strongest variables for separation of subgroups. CONCLUSIONS: Enhanced sensitivity to depolarisation and to halothane appear to be the primary, mutually reinforcing and phenotype-defining defects of halothane-hypersensitive patients with malignant hyperthermia susceptibility.

Muscle calcium stress cleaves junctophilin1, unleashing a gene regulatory program predicted to correct glucose dysregulation.

Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3ß - a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing CaV1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3ß and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44.

A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus, τ2 was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle, τ2 was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). From τ2 distributions, it follows that an FDB fiber with τ2 > 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

Quantification of the calcium signaling deficit in muscles devoid of triadin.

Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The "hump" phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.

Real-World Characteristics and Outcome of Patients Treated With Single-Agent Ibrutinib for Chronic Lymphocytic Leukemia in Spain (IBRORS-LLC Study).

BACKGROUND: Ibrutinib demonstrated remarkable efficacy and favorable tolerability in patients with untreated or relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL), including those with high-risk genetic alterations. The IBRORS-CLL study assessed the characteristics, clinical management and outcome of CLL patients receiving ibrutinib in routine clinical practice in Spain. PATIENTS: Observational, retrospective, multicenter study in CLL patients who started single-agent ibrutinib as first-line treatment or at first or second relapse between January 2016 and January 2019. RESULTS: A total of 269 patients were included (median age: 70.9 years; cardiovascular comorbidity: 55.4%, including hypertension [47.6%] and atrial fibrillation [AF] [7.1%]). Overall, 96.7% and 69% of patients underwent molecular testing for del(17p)/TP53 mutation and IGHV mutation status. High-risk genetic features included unmutated IGHV (79%) and del(17p)/TP53 mutation (first-line: 66.3%; second-line: 23.1%). Overall, 84 (31.2%) patients received ibrutinib as first-line treatment, and it was used as second- and third-line therapy in 121 (45.0%) and 64 (23.8%) patients. The median progression-free survival and overall survival were not reached irrespective of del(17p)/TP53, or unmutated IGHV. Common grade ≥3 adverse events were infections (12.2%) and bleeding (3%). Grade ≥3 AF occurred in 1.5% of patients. CONCLUSION: This real-world study shows that single-agent ibrutinib is an effective therapy for CLL, regardless of age and high-risk molecular features, consistent with clinical trials. Additionally, single-agent ibrutinib was well tolerated, with a low rate of cardiovascular events. This study also emphasized a high molecular testing rate of del(17p)/TP53 mutation and IGHV mutation status in clinical practice according to guideline recommendations.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction.

KEY POINTS: Accumulation of inorganic phosphate (Pi ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established.  We showed that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR .  Pre-treatment of permeabilized myofibres with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi .  The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature. ABSTRACT: Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

De la medicina narrativa a los cuidados humanizados: El hospital El Cruce en pandemia

Este libro intenta compartir los aportes técnicos de los enfoques con las vivencias emocionales recogidas en múltiples relatos. Contiene descripciones rigurosas de las dos disciplinas que confluyen, la medicina narrativa y los cuidados humanizados, así como de las medidas adoptadas en la organización del hospital y la asistencia. Suma a esos contenidos el relato de la experiencia en todo el enfoque humanístico de la crisis, de la reflexión grupal en los talleres, las estrategias adoptadas para la resolución de problemas complejos como las visitas, el aislamiento y los cuidados del final de la vida. En cada tema, aporta textos comprometidos y personales de los actores y diversos profesionales de la salud que comparten sus emociones y conmociones en la circunstancia extrema de la pandemia. (AU)

A multi-dimensional analysis of genotype-phenotype discordance in malignant hyperthermia susceptibility.

BACKGROUND: Malignant hyperthermia (MH) susceptibility is an inherited condition, diagnosed either by the presence of a pathogenic genetic variant or by in vitro caffeine-halothane contracture testing. Through a multi-dimensional approach, we describe the implications of discordance between genetic and in vitro test results in a patient with a family history of possible MH. METHODS: The patient, whose brother had a possible MH reaction, underwent the caffeine-halothane contracture test (CHCT) according to the North American MH Group protocol. Screening of the complete RYR1 and CACNA1S transcripts was done using Sanger sequencing. Additional functional analyses included skinned myofibre calcium-induced calcium release sensitivity, calcium signalling assays in cultured myotubes, and in silico evaluation of the effect of any genetic variants on their chemical environment. RESULTS: The patient's CHCT result was negative but she carried an RYR1 variant c.1209C>G, p.Ile403Met, that is listed as pathogenic by the European Malignant Hyperthermia Group. Functional tests indicated a gain-of-function effect with a weak impact, and the variant was predicted to affect the folding stability of the 3D structure of the RyR1 protein. Based on American College of Medical Genetics and Genomics/Association of Molecular Pathology guidelines, this variant would be characterised as a variant of uncertain significance. CONCLUSIONS: Available data do not confirm or exclude an increased risk of MH for this patient. Further research is needed to correlate RyR1 functional assays, including the current gold standard testing for MH susceptibility, with clinical phenotypes. The pathogenicity of genetic variants associated with MH susceptibility should be re-evaluated.

Intracellular calcium leak lowers glucose storage in human muscle, promoting hyperglycemia and diabetes.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca2+]cytosol. 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GPa), glycogen synthase (GSa) - respectively activated and inactivated by phosphorylation - and their Ca2+-dependent kinase (PhK), are elevated in microsomal extracts from MHS patients' muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca2+]cytosol, increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GPa at sarcoplasmic reticulum (SR) junctional cisternae, and apo-GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca2+]cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients' hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links.

Animals and humans move by contracting the skeletal muscles attached to their bones. These muscles take up a type of sugar called glucose from food and use it to fuel contractions or store it for later in the form of glycogen. If muscles fail to use glucose it can lead to excessive sugar levels in the blood and a condition called diabetes. Within muscle cells are stores of calcium that signal the muscle to contract. Changes in calcium levels enhance the uptake of glucose that fuel these contractions. However, variations in calcium have also been linked to diabetes, and it remained unclear when and how these 'signals' become harmful. People with a condition called malignant hyperthermia susceptibility (MHS for short) have genetic mutations that allow calcium to leak out from these stores. This condition may result in excessive contractions causing the muscle to over-heat, become rigid and break down, which can lead to death if left untreated. A clinical study in 2019 found that out of hundreds of patients who had MHS, nearly half had high blood sugar and were likely to develop diabetes. Now, Tammineni et al. ­ including some of the researchers involved in the 2019 study ­ have set out to find why calcium leaks lead to elevated blood sugar levels. The experiments showed that enzymes that help convert glycogen to glucose are more active in patients with MHS, and found in different locations inside muscle cells. Whereas the enzymes that change glucose into glycogen are less active. This slows down the conversion of glucose into glycogen for storage and speeds up the breakdown of glycogen into glucose. Patients with MHS also had fewer molecules that transport glucose into muscle cells and stored less glycogen. These changes imply that less glucose is being removed from the blood. Next, Tammineni et al. used a microscopy technique that is able to distinguish finely separated objects with a precision not reached before in living muscle. This revealed that when the activity of the enzyme that breaks down glycogen increased, it moved next to the calcium store. This effect was also observed in the muscle cells of MHS patients that leaked calcium from their stores. Taken together, these observations may explain why patients with MHS have high levels of sugar in their blood. These findings suggest that MHS may start decades before developing diabetes and blood sugar levels in these patients should be regularly monitored. Future studies should investigate whether drugs that block calcium from leaking may help prevent high blood sugar in patients with MHS or other conditions that cause a similar calcium leak.

Abordaje transfibular modificado para fracturas del platillo tibial posterolateral. Serie de Casos / Modified transfibular approach for posterolateral tibial plateau fractures. Case Series

Introducción Las fracturas del platillo tibial posterolateral exigen un abordaje que permita una buena visualización de la superficie articular para su restitución anatómica, restablecimiento de la altura y un espacio adecuado para la aplicación del material de osteosíntesis y fijación estable. Hay diversos abordajes descritos en la literatura que no proporcionan la visualización deseada o conllevan múltiples limitaciones y/o complicaciones. El propósito de este estudio es describir una modificación a la técnica quirúrgica de la osteotomía de la fíbula para el manejo de las fracturas de platillo tibial posterolateral. Materiales y métodos Se presentan los resultados clínicos de una serie de casos retrospectiva de 15 pacientes con fracturas de platillo tibial posterolateral que al momento del estudio tenían un seguimiento promedio de 6 meses. Resultados Los resultados funcionales en escala de Lysholm fueron excelentes en 3 pacientes, buenos en 8 y regulares en 4. En todos los pacientes se logró una reducción satisfactoria de la superficie articular de la tibia, sin pérdida de altura de la misma, con alineación adecuada, sin síntomas de inestabilidad, todos con consolidación de la fíbula que no retrasó la rehabilitación, no hubo lesiones del nervio peroneal ni problemas con la piel; se presentó un caso de infección superficial que se manejó con antibióticos orales sin complicaciones. Discusión El abordaje descrito facilita una amplia exposición de la superficie articular, es técnicamente fácil y reproducible, permite la reducción y fijación necesaria, con menor riesgo de complicaciones, con la cual se conserva el aporte vascular de la tibia proximal, se evita el daño a la articulación tibiofibular proximal y se propicia una mayor área de consolidación de la osteotomía. Ésta técnica puede utilizarse sola o en combinación con otros abordajes, para fracturas agudas o crónicas con mala unión. Consideramos que el abordaje con la técnica descrita es una alternativa para el manejo de las fracturas posterolaterales, con resultados alentadores.

Background Fractures of the posterolateral tibial plateau require an approach that allows a good visualization of the articular surface for its anatomical restitution, restoration of height and an adequate space for the application of osteosynthesis material and stable fixation. There are several approaches described in the literature that do not provide the desired visualization or involve multiple limitations and / or complications. The purpose of this study is to describe a modification to the surgical technique of the fibula osteotomy for the management of posterolateral tibial plate fractures. Methods We present the clinical results of a retrospective case series of 15 patients with posterolateral tibial plate fractures that at the time of the study had an average follow-up of 6 months. Results Functional results in Lysholm scale were excellent in 3 patients, good in 8 and regular in 4. Good results were obtained in all patients with a reduction of the tibia articular surface, without loss of height of the same, with alignment adequate, without symptoms of instability, all with consolidation of the fibula that did not delay rehabilitation, there were no peroneal nerve injuries or problems with the skin; There was a case of superficial infection that was managed with oral antibiotics without complications. Discussion The described approach facilitates a broad exposure of the articular surface, is technically easy and reproducible, allows the necessary reduction and fixation, with a lower risk of complications, with which the vascular supply of the proximal tibia is conserved, the damage is avoided to the proximal tibiofibular joint and a greater area of consolidation of the osteotomy is favored. This technique can be used alone or in combination with other approaches, for acute or chronic fractures with poor union. We consider that the approach with the described technique is an alternative for the management of posterolateral fractures, with encouraging results.

The binding interactions that maintain excitation-contraction coupling junctions in skeletal muscle.

Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca2+ (CaV1.1) channels, found in tubules or plasmalemma, form symmetric complexes called CaV tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, CaV tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor-lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between CaVs and RYRs and test this hypothesis using a quantitative model of the energies that govern CaV1.1-RYR1 binding, which are assumed to depend on number and location of bound CaVs. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by CaVs, so that CaVs positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two CaVs bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.

Calcium-induced release of calcium in muscle: 50 years of work and the emerging consensus.

Ryanodine-sensitive intracellular Ca2+ channels (RyRs) open upon binding Ca2+ at cytosolic-facing sites. This results in concerted, self-reinforcing opening of RyRs clustered in specialized regions on the membranes of Ca2+ storage organelles (endoplasmic reticulum and sarcoplasmic reticulum), a process that produces Ca2+-induced Ca2+ release (CICR). The process is optimized to achieve large but brief and localized increases in cytosolic Ca2+ concentration, a feature now believed to be critical for encoding the multiplicity of signals conveyed by this ion. In this paper, I trace the path of research that led to a consensus on the physiological significance of CICR in skeletal muscle, beginning with its discovery. I focus on the approaches that were developed to quantify the contribution of CICR to the Ca2+ increase that results in contraction, as opposed to the flux activated directly by membrane depolarization (depolarization-induced Ca2+ release [DICR]). Although the emerging consensus is that CICR plays an important role alongside DICR in most taxa, its contribution in most mammalian muscles appears to be limited to embryogenesis. Finally, I survey the relevance of CICR, confirmed or plausible, to pathogenesis as well as the multiple questions about activation of release channels that remain unanswered after 50 years.

The voltage sensor of excitation-contraction coupling in mammals: Inactivation and interaction with Ca2.

In skeletal muscle, the four-helix voltage-sensing modules (VSMs) of CaV1.1 calcium channels simultaneously gate two Ca2+ pathways: the CaV1.1 pore itself and the RyR1 calcium release channel in the sarcoplasmic reticulum. Here, to gain insight into the mechanism by which VSMs gate RyR1, we quantify intramembrane charge movement associated with VSM activation (sensing current) and gated Ca2+ release flux in single muscle cells of mice and rats. As found for most four-helix VSMs, upon sustained depolarization, rodent VSMs lose the ability to activate Ca2+ release channels opening; their properties change from a functionally capable mode, in which the mobile sensor charge is called charge 1, to an inactivated mode, charge 2, with a voltage dependence shifted toward more negative voltages. We find that charge 2 is promoted and Ca2+ release inactivated when resting, well-polarized muscle cells are exposed to low extracellular [Ca2+] and that the opposite occurs in high [Ca2+]. It follows that murine VSMs are partly inactivated at rest, which establishes the reduced availability of voltage sensing as a pathogenic mechanism in disorders of calcemia. We additionally find that the degree of resting inactivation is significantly different in two mouse strains, which underscores the variability of voltage sensor properties and their vulnerability to environmental conditions. Our studies reveal that the resting and activated states of VSMs are equally favored by extracellular Ca2+ Promotion by an extracellular species of two states of the VSM that differ in the conformation of the activation gate requires the existence of a second gate, inactivation, topologically extracellular and therefore accessible from outside regardless of the activation state.

Characteristics and management of primary and other immune thrombocytopenias: Spanish registry study.

BACKGROUND: The natural history and its modulation by treatments administered for immune thrombocytopenia (ITP) in the clinical practice remains unknown. In addition, little information is available on the characteristics and management of ITP in Spain. METHODS: We conducted an observational, multicenter, registry in 70 Hematology Services from Spain between 2009 and 2011, which included children from 2 months of age and adults with primary ITP or another ITP diagnosed within the last 6 months (platelet count [PC] < 100 × 109/l). Patients were followed-up at 6 and 12 months. RESULTS: 484 patients were included (median [Q1, Q3] age 52 [29,74] years, 87.6% adults), 56% women, 10.5% with secondary ITP. Median (Q1, Q3) PC at diagnosis was 12 × 109/l (4, 32); 72% of patients had bleeding symptoms (62% cutaneous bleeding, 29% oral cavity bleeding, 18% epistaxis). 81% of patients with primary ITP received first-line treatment, mainly with corticosteroids (>6 weeks in 59% of cases), either alone (41%) or associated with intravenous immunoglobulin (33%). The response (≥30 × 109/L) to first-line treatment was 92%. A total of 19% of patients received second-line treatment and 6% additional treatments. At 12 months, 74% of primary ITP patients maintained a PC ≥ 100 × 109/L in absence of treatment (10% still had hemorrhagic manifestations). CONCLUSIONS: Characteristics of Spanish ITP patients are comparable to those from other countries. Although a high response rate to first-line treatments is observed, at 1 year, the disease persists in around one quarter of patients. Overall therapeutic management in Spain conforms to current recommendations, except for an excessive duration of corticosteroids therapy.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle.

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR--measured with a calsequestrin-fused biosensor--was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.

The HUGE formula (hematocrit, urea, gender) for screening for chronic kidney disease in elderly patients: a study of diagnostic accuracy.

Chronically reduced glomerular filtration rate (GFR) in old people does not always mean that they suffer from chronic kidney disease (CKD) since their GFR can just be reduced by aging. The HUGE equation has been recently described and validated in Spain for screening CKD without taking into account the patient's GFR value. This equation is based on patient's hematocrit, plasma urea levels and gender. The present study documented that the HUGE equation had and acceptable performance for screening CKD in elderly Argentine patients.

Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle.

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca(2+)-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic "guiderail"-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca(2+) concentrations, and proper glycosylation, in turn, guarantees both effective Ca(2+) storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.

Alterations in zebrafish development induced by simvastatin: Comprehensive morphological and physiological study, focusing on muscle.

The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin.