Long-term efficacy and safety of cardiac genome editing for catecholaminergic polymorphic ventricular tachycardia.

Introduction: Heterozygous autosomal-dominant single nucleotide variants in RYR2 account for 60% of cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia disorder associated with high mortality rates. CRISPR/Cas9-mediated genome editing is a promising therapeutic approach that can permanently cure the disease by removing the mutant RYR2 allele. However, the safety and long-term efficacy of this strategy have not been established in a relevant disease model. Aim: The purpose of this study was to assess whether adeno-associated virus type-9 (AAV9)-mediated somatic genome editing could prevent ventricular arrhythmias by removal of the mutant allele in mice that are heterozygous for Ryr2 variant p.Arg176Gln (R176Q/+). Methods and Results: Guide RNA and SaCas9 were delivered using AAV9 vectors injected subcutaneously in 10-day-old mice. At 6 weeks after injection, R176Q/+ mice had a 100% reduction in ventricular arrhythmias compared to controls. When aged to 12 months, injected R176Q/+ mice maintained a 100% reduction in arrhythmia induction. Deep RNA sequencing revealed the formation of insertions/deletions at the target site with minimal off-target editing on the wild-type allele. Consequently, CRISPR/SaCas9 editing resulted in a 45% reduction of total Ryr2 mRNA and a 38% reduction in RyR2 protein. Genome editing was well tolerated based on serial echocardiography, revealing unaltered cardiac function and structure up to 12 months after AAV9 injection. Conclusion: Taken together, AAV9-mediated CRISPR/Cas9 genome editing could efficiently disrupt the mutant Ryr2 allele, preventing lethal arrhythmias while preserving normal cardiac function in the R176Q/+ mouse model of CPVT.

Phosphorylation of RyR2 simultaneously expands the dyad and rearranges the tetramers.

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately, making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. Here, we used the ß-agonist isoproterenol and mice homozygous for one of the following clinically relevant mutations: S2030A, S2808A, S2814A, or S2814D. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that the S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers, suggesting a direct link between the phosphorylation state of the tetramer and its microarchitecture. S2808A and S2814A mutant mice, as well as wild types, had significant expansions of their dyads in response to isoproterenol, while S2030A mutants did not. In agreement with functional data from these mutants, S2030 and S2808 were necessary for a complete ß-adrenergic response, unlike S2814 mutants. Additionally, all mutants had unique effects on the organization of their tetramer arrays. Lastly, the correlation of structural with functional changes suggests that tetramer-tetramer contacts play an important functional role. We thus conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a ß-adrenergic receptor agonist.

Intracellular calcium mishandling leads to cardiac dysfunction and ventricular arrhythmias in a mouse model of propionic acidemia.

Propionic acidemia (PA) is a rare metabolic disease associated with mutations in genes encoding the α and ß subunits of the enzyme propionyl-CoA carboxylase. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species production and oxidative damage, which have been associated with the disease pathophysiology. Clinical symptoms are heterogeneous and include cardiac complications, mainly cardiac dysfunction and arrhythmias, which are recognized as one of the major life-threatening manifestations in patients. We aimed to investigate the molecular mechanisms underlying the cardiac phenotype using a hypomorphic mouse model (Pcca-/-(A138T)) that recapitulates some biochemical and clinical characteristics of PA. We demonstrate that Pcca-/-(A138T) mice present with depressed cardiac function along with impaired cell contractility when compared to the wild-type mice. Cardiac dysfunction in Pcca-/-(A138T) mice was associated with lower systolic Ca2+ release ([Ca2+]i transients), impairment in the sarcoplasmic reticulum (SR) Ca2+ load and decreased Ca2+ re-uptake by SR-Ca2+ ATPase (SERCA2a). These functional changes correlated well with the depressed activity of SERCA2a, the elevated ROS levels and SERCA2a oxidation rate in cardiomyocytes isolated from Pcca-/-(A138T) mice. In addition, decreased SR-Ca2+ load in Pcca-/-(A138T) cardiomyocytes was associated with increased diastolic Ca2+ release. The increase in Ca2+ sparks, Ca2+ waves and spontaneous [Ca2+]i transients in Pcca-/-(A138T) cardiomyocytes could be responsible for the induction of ventricular arrhythmias detected in these mice. Overall, our results uncover the role of impaired Ca2+ handling in arrhythmias and cardiac dysfunction in PA, and identify new targets for the development of therapeutic approaches for this devastating metabolic disease.

Optimización de la farmacoterapia en un hospital de Traumatología / Optimisation of pharmacotherapy in a trauma centre

Objetivo Evaluar los resultados de la implantación de un programa de atención farmacéutica dirigido a optimizar el tratamiento farmacoterapéutico individualizado en un hospital de Traumatología con historia clínica informatizada (HCI) y sistema integral de dispensación individualizada de medicamentos (SIDIM).Métodos Estudio retrospectivo observacional de 3 años de duración (2007-2009). Se realizó un seguimiento diario del tratamiento farmacoterapéutico de los pacientes ingresados en unidades de hospitalización con SIDIM. Mediante el registro en un documento normalizado, se clasificaron los problemas relacionados con los medicamentos (PRM) y/o errores de medicación (EM) identificados, así como las intervenciones farmacéuticas realizadas de acuerdo con la idoneidad y el grado de aceptación de las mismas. Para la identificación de pacientes con oportunidades de mejora en su farmacoterapia (PRM y/o EM) se empleó el método IASER®.Resultados Se realizaron 1.971 intervenciones farmacéuticas (IF) tras revisar 124.336 líneas de tratamiento correspondientes a 12 IF por cada 100 pacientes. La prevalencia de pacientes con PRM fue del 12% distribuidos de la siguiente manera: 50,66% categorizados en seguridad, 22,98% en indicación, 12,23% en efectividad y 14,13% en adherencia. Los grupos fármacológicos principalmente implicados fueron: antiinfecciosos (29%), fármacos para el aparato locomotor (..) (AU)

Objective To evaluate the results for implementing a pharmaceutical care programme aimed at optimising personalised pharmacotherapeutic treatment in a Trauma Centre with electronic medical records (EMR) and an integral system for personalised medication dispensing (ISPMD).Method A three-year observational, retrospective study (2007-2009). On a daily basis, we checked the pharmaceutical treatment of patients admitted to hospital units with ISPMD. The medication-related problems (MRP) and medication errors (ME) were identified and classified by recording them on a standardised document. We also recorded data on the Pharmaceutical Interventions performed in accordance with fitness and level of acceptance. We used the laser® method to identify patients with pharmacotherapy improvement opportunities (MRP and/or ME).Results One thousand nine-hundred and seventy-one pharmaceutical interventions (PI) were found after having reviewed 124 336 treatment lines, resulting in 12 PI for every 100 patients. The prevalence of patients with MRP was 12%, distributed as such: 50.66% were safety-related, 22.98% indication-related, 12.23% effectiveness-related and 14.13% adherence-related. The main drug groups involved were: anti-infectious agents (29%), drugs for the musculoskeletal system (21%), drugs for blood and haematopoietic organs (12%), and drugs for the nervous system (11%). The active ingredient that required most PI in 2007 was dexketoprofen (15.6%), followed by ketorolac (12.4%). In 2008, it was dexketoprofen (22.0%) followed by gentamicin (7.3%), and in 2009 enoxaparin (19.0%) followed by dexketoprofen (14.3%). The origin of MRP was due to ME in 91% of cases in 2007and 81% in 2008, decreasing (..) (AU)

[Optimisation of pharmacotherapy in a trauma centre]. / Optimización de la farmacoterapia en un hospital de Traumatología.

OBJECTIVE: To evaluate the results for implementing a pharmaceutical care programme aimed at optimising personalised pharmacotherapeutic treatment in a Trauma Centre with electronic medical records (EMR) and an integral system for personalised medication dispensing (ISPMD). METHOD: A three-year observational, retrospective study (2007-2009). On a daily basis, we checked the pharmaceutical treatment of patients admitted to hospital units with ISPMD. The medication-related problems (MRP) and medication errors (ME) were identified and classified by recording them on a standardised document. We also recorded data on the Pharmaceutical Interventions performed in accordance with fitness and level of acceptance. We used the laser method to identify patients with pharmacotherapy improvement opportunities (MRP and/or ME). RESULTS: One thousand nine-hundred and seventy-one pharmaceutical interventions (PI) were found after having reviewed 124 336 treatment lines, resulting in 12 PI for every 100 patients. The prevalence of patients with MRP was 12%, distributed as such: 50.66% were safety-related, 22.98% indication-related, 12.23% effectiveness-related and 14.13% adherence-related. The main drug groups involved were: anti-infectious agents (29%), drugs for the musculoskeletal system (21%), drugs for blood and haematopoietic organs (12%), and drugs for the nervous system (11%). The active ingredient that required most PI in 2007 was dexketoprofen (15.6%), followed by ketorolac (12.4%). In 2008, it was dexketoprofen (22.0%) followed by gentamicin (7.3%), and in 2009 enoxaparin (19.0%) followed by dexketoprofen (14.3%). The origin of MRP was due to ME in 91% of cases in 2007 and 81% in 2008, decreasing to 53% in 2009. PI fitness, as percentages (CI 95%) were considered: Important PI [30.29 (10.19-49.95)]; Very important PI [38.36 (35.45-73)]; Acceptable PI [82.10 (52.28-111.10)]. CONCLUSIONS: Optimising personalised pharmacotherapeutic treatment by implementing an interdisciplinary Pharmaceutical Care programme promotes team work, and as a result improves rational and safe medication dispensing.

The effect of temperature on food emulsifiers at fluid-fluid interfaces.

Heat-induced interfacial aggregation of a whey protein isolate (WPI) with a high content of beta-lactoglobulin (>92%), previously adsorbed at the oil-water interface, was studied by means of interfacial dynamic characteristics performed in an automatic drop tensiometer. Protein concentration in aqueous bulk phase ranging between 1x10(-1) and 1x10(-5) % wt/wt was studied as a variable. The experiments were carried out at temperatures ranging from 20-80 degrees C with different thermal regimes. During the heating period, competition exists between the effect of temperature on the film fluidity and the increase in mechanical properties associated with the interfacial gelation process. Interfacial crystallisation of food polar lipids (monopalmitin, monoolein, and monolaurin) previously adsorbed at the oil-water interface, was studied by interfacial dynamic characteristics (interfacial tension and surface dilational properties). The temperature, ranging between 40 and 2 degrees C, and the lipid concentration in aqueous oil phase, ranging between 1x10(-2) and 1x10(-4) % wt/wt, were studied as variables. Significant changes in interfacial dynamic characteristics associated with interfacial lipid crystallisation were observed as a function of lipid concentration in the bulk phase. Interfacial crystallisation of food polar lipids (monopalmitin, monoolein, and monolaurin) at the air-water interface, was studied by pi-A isotherms performed in a Langmuir trough coupled with Brewster angle microscopy (BAM). A condensation in monoglyceride monolayers towards lower molecular area was observed as the temperature decreased. This effect was attributed to lipid crystallisation at lower temperatures. BAM images corroborated the effect of temperature on the monolayer structure, as a function of the monoglyceride type.

Protein-lipid interactions at the oil-water interface.

The distribution of proteins and lipids in food emulsions and foams is determined by competitive and cooperative adsorption between the two types of emulsifiers at the fluid-fluid interfaces, and by the nature of protein-lipid interactions, both at the interface and in the bulk phase. The existence of protein-lipid interactions can have a pronounced impact on the surface rheological properties of these systems. Therefore, these results are of practical importance for food emulsion formulation, texture, and stability. In this study, the existence of protein-lipid interactions at the interface was determined by surface dynamic properties (interfacial tension and surface dilational modulus). Systematic experimental data on surface dynamic properties, as a function of time and at long-term adsorption, for protein (whey protein isolate (WPI)), lipids (monoglycerides), and protein-lipid mixed films at the oil-water interface were measured in an automated drop tensiometer. The dynamic behaviour of protein+lipid mixed films depends on the adsorption time, the lipid and the protein/lipid ratio in a rather complicated manner. The protein determined the interfacial characteristics of the mixed film as the protein at WPI>/=10(-2)% wt/wt saturated the film, no matter what the concentration of the lipid. However, there exists a competitive or cooperative adsorption of the emulsifier (WPI and monoglycerides), as the concentration of protein in the bulk phase is far lower than that for interfacial saturation.

[Survival in cancer of the breast in Zaragoza (1960-1990) in relation to age, clinical stage and period of time of the diagnosis]. / Supervivencia del cáncer de mama en Zaragoza (1960-1990) en relación con la edad, estadio clínico y período de tiempo del diagnóstico.

BACKGROUND: The characteristics of a population based series of 3,066 women diagnosed with breast cancer collected by the Cancer Registry of Zaragoza, Spain from 1960 to 1990 are herein described. Gross short and long term survival, as well as specific survival were estimated according to age at diagnosis, tumor stage and the period in which the patient was diagnosed. METHODS: Every patient was followed up to verification of death or to the latest information available up to January 1, 1991. Diagnosis specified in writing in the clinical history and support by surgical or anatomopathologic reports were required. The data concerning place and site of residence and the vital status of the cases were verified by the municipal and civil registries, death certificates and burial registries. The survival curves were estimated by the Kaplan-Meier method, and short and long term survival, by age and tumor stage at diagnosis and the diagnostic period were also evaluated. RESULTS: Gross survival was estimated as 89.1% in the first year, 50.9% at 5 years, and 34.7%, 28.4% and 20.0% at 10, 15, 20 and 25 years, respectively. Survival at 5 years according to TNM clinical stage classification (UICC-AJC 1978) was 90% for stage I, 69.5% for stage II, 44.6% for stage III and 20.6% for stage IV. The cases diagnosed between 1980 and 1990 presented better survival than those previously diagnosed. The differences were statistically significant in all the cases (p < 0.001, log-rank test). CONCLUSIONS: A slight improvement has been observed in the survival of women diagnosed with breast cancer in Zaragoza, Spain during the study period. Despite of that fact, the survival rates were worse than those observed in other countries with similar socioeconomic development.