Calmodulin Binding to the 3614–3643 Region of RyR1 Is Not Essential for Excitation–Contraction Coupling in Skeletal Myotubes

Calmodulin is a ubiquitous Ca2+ binding protein that modulates the in vitro activity of the skeletal muscle ryanodine receptor (RyR1). Residues 3614–3643 of RyR1 comprise the CaM binding domain and mutations within this region result in a loss of both high-affinity Ca2+-bound calmodulin (CaCaM) and Ca2+-free CaM (apoCaM) binding (L3624D) or only CaCaM binding (W3620A). To investigate the functional role of CaM binding to this region of RyR1 in intact skeletal muscle, we compared the ability of RyR1, L3624D, and W3620A to restore excitation–contraction (EC) coupling after expression in RyR1-deficient (dyspedic) myotubes. W3620A-expressing cells responded normally to 10 mM caffeine and 500 μM 4-chloro-m-cresol (4-cmc). Interestingly, L3624D-expressing cells displayed a bimodal response to caffeine, with a large proportion of cells (∼44%) showing a greatly attenuated response to caffeine. However, high and low caffeine-responsive L3624D-expressing myotubes exhibited Ca2+ transients of similar magnitude after activation by 4-cmc (500 μM) and electrical stimulation. Expression of either L3624D or W3620A in dyspedic myotubes restored both L-type Ca2+ currents (retrograde coupling) and voltage-gated SR Ca2+ release (orthograde coupling) to a similar degree as that observed for wild-type RyR1, although L-current density was somewhat larger and activated at more hyperpolarized potentials in W3620A-expressing myotubes. The results indicate that CaM binding to the 3614–3643 region of RyR1 is not essential for voltage sensor activation of RyR1

Development and Pretesting of a Questionnaire to Assess Patient Experiences and Satisfaction with Medications (PESaM Questionnaire)

Background: The aim of this study was to develop, together with the Lung Foundation Netherlands and Dutch Kidney Patients Association, patients and clinicians, a measure to evaluate patient experiences with the orphan drugs pirfenidone (for idiopathic pulmonary fibrosis [IPF]) and eculizumab (for atypical haemolytic uraemic syndrome [aHUS]), as well as a generic measure of patient experiences and satisfaction with medications. Methods: Development of the Patient Experiences and Satisfaction with Medications (PESaM) questionnaire consisted of four phases: literature review (phase I); focus groups and individual patient interviews (phase II); item generation (phase III); and face and content validity testing (phase IV). Literature review aimed to identify existing disease-specific and generic patient experience measures to provide guidance on the domains of medication use relevant to patients, the number of items and type of response categories, and to generate an initial pool of items. Subsequent focus groups and patient interviews were conducted to gain insight into the perceived effectiveness of the therapies, the bur

Validity of the Patient Experiences and Satisfaction with Medications (PESaM) Questionnaire

Background: This study assessed the validity and reliability of the generic module of the recently developed Patient Experiences and Satisfaction with Medications (PESaM) questionnaire in a sample of patients in the Netherlands. Methods: The generic module of the PESaM questionnaire consists of 18 items related to the domains effectiveness, side effects and ease of use of medications. It assesses patients’ experiences regarding the impact of the medication on daily life, health and satisfaction. In 2017, the PESaM questionnaire was sent out to idiopathic pulmonary fibrosis patients using pirfenidone or nintedanib, atypical haemolytic uraemic syndrome patients receiving eculizumab and patients using tacrolimus after kidney transplantation. Mean scores for each domain were calculated applying a scoring algorithm. Construct validity and reliability were assessed using recommended methods. Results: 188 participants completed the generic module, of whom 48% used pirfenidone, 36% nintedanib, 11% tacrolimus and 5% eculizumab. The generic module has good structural properties. Internal consistency values of the domains were satisfactory (i.e. Cronbach’s coefficient alpha above 0.7). Confirmatory factor analysis provided further evidence for construct validity, with good convergent and discriminant validity. The PESaM questionnaire also showed different scores for patients using different medications, in line with expectations, and was therefore able to differentiate between patient groups. Test–retest reliability of the items and domains were rated as moderate to fair (i.e. intraclass coefficients ranged between 0.18 and 0.76). Conclusions: The PESaM questionnaire is a unique patient-reported outcome measure evaluating patient experiences and satisfaction with medications. It has been developed in conjunction with patients, ensuring coverage of domains and issues relevant from the patient’s perspective. This study has shown promising validity of the generic module of the PESaM questionnaire. Further research is recommended to assess reliability in greater detail as well as the responsiveness of the measure. Trial registration: The study

A 3D active shape model driven by fuzzy inference : application to cardiac CT and MR

Abstract—Manual quantitative analysis of cardiac left ventricular function using Multislice CT and MR is arduous because of the large data volume. In this paper, we present a 3-D active shape model (ASM) for semiautomatic segmentation of cardiac CT and MRvolumes, without the requirement of retraining the underlying statistical shape model. A fuzzy c-means based fuzzy inference system was incorporated into the model. Thus, relative gray-level differences instead of absolute gray values were used for classification of 3-D regions of interest (ROIs), removing the necessity of training different models for different modalities/acquisition protocols. The 3-D ASM was evaluated using 25 CT and 15 MR datasets. Automatically generated contours were compared to expert contours in 100 locations. For CT, 82.4% of epicardial contours and 74.1% of endocardial contours had a maximum error of 5 mm along 95% of the contour arc length. For MR, those numbers were 93.2% (epicardium) and 91.4% (endocardium). Volume regression analysis revealed good linear correlations between manual and semiautomatic volumes, r2 = 0.98. This study shows that the fuzzy inference 3-D ASM is a robust promising instrument for semiautomatic cardiac left ventricle segmentation.Without retraining its statistical shape component, it is applicable to routinely acquired CT and MR studies

Magnetically navigated percutaneous coronary intervention in distal and/or complex lesions may improve procedural outcome and material consumption

Aims: Comparison of magnetic guidewire navigation in percutaneous coronary intervention (magnetic PCI) across distal and /or complex lesions versus conventional navigation (conventional PCI). Methods and results: Forty-seven consecutive patients (age 61±10yr) undergoing elective single vessel magnetic PCI for distal and/or complex lesions were matched by age and lesion location with 45 patients undergoing conventional PCI (age 63±10yr). Technical success rate was defined

Follow-up of Contacts of Middle East Respiratory Syndrome Coronavirus–Infected Returning Travelers, the Netherlands, 2014

Notification of 2 imported cases of infection with Middle East respiratory syndrome coronavirus in the Netherlands triggered comprehensive monitoring of contacts. Observed low rates of virus transmission and the psychological effect of contact monitoring indicate that thoughtful assessment of close contacts is prudent and must be guided by clinical and epidemiologic risk factors

Absorption and metabolism of volatile fatty acids by rumen and omasum Absorção e metabolismo de ácidos graxos voláteis pelo rúmen e omaso

Volatile fatty acids (VFA) absorption and metabolic capacity of rumen and omasum were compared, in vitro. Fragments of rumen wall and omasum laminae were taken from eight adult crossbred bovines. An isolated fragment of the mucosa was fitted in a tissue diffusion chamber. Valeric acid and CrEDTA were added to ruminal fluid and placed on the mucosal side and buffer solution was placed on the serosal side. Fractional absorption rates were measured by exponential VFA:Cr ratio decay over time. Metabolism rate was determined as the difference between VFA absorbed and VFA which appeared on the serosal side over time. Mitotic index was higher in omasum (0.52%) than in rumen epithelium (0.28%). VFA fractional absorption rate was higher in omasum (4.6%/h.cm²) than in rumen (0.4%/h.cm²). Acetate, propionate, butyrate, and valerate showed similar fractional absorption rates in both fragments. Percentage of metabolized acetate and propionate was lower than butyrate and valerate in both stomach compartments. In the rumen, individual VFA metabolism rates were similar (mean of 7.7 , but in the omasum, valerate (90.0 was more metabolized than butyrate (59.6 propionate (69.8 and acetate (51.7 . Correlation between VFA metabolism and mitotic index was positive in the rumen and in the omasum. In conclusion, VFA metabolism and absorption potential per surface of the omasum is higher than that of the rumen. Variations on rumen and omasum absorption capacities occur in the same way, and there are indications that factors capable of stimulating rumen wall proliferation are similarly capable of stimulating omasum walls.<br>A capacidade de absorção e metabolismo de ácidos graxos voláteis (AGV) pelo rúmen e omaso foi comparada, in vitro. Fragmentos da parede do rúmen e das lâminas do omaso foram coletados de oito bovinos mestiços adultos. Um fragmento isolado da mucosa foi colocado em uma câmara de difusão tecidual. Ácido valérico e CrEDTA foram adicionados ao fluido ruminal e colocados no compartimento da câmara voltados para a mucosa e uma solução tampão foi colocada no compartimento voltado para a serosa. As taxas fracionais de absorção foram medidas pela queda exponencial da relação VFA:Cr ao longo do tempo. A taxa de metabolismo foi determinada pela diferença entre a quantidade de AGV absorvida e a detectada no compartimento serosal da câmara. O índice mitótico foi mais alto no epitélio do omaso (0.52%) do que no do rúmen (0.28%), bem como a taxa fracional de absorção, 4.6%/h.cm² e 0.4%/h.cm², respectivamente. Acetato, propionato, butirato e valerato tiveram taxas fracionais de absorções similares em ambos os compartimentos. As porcentagens do acetato e do propionato metabolizados foram mais baixas do que a do butirato e valerato em ambos os compartimentos. No rúmen, a taxa metabólica individual dos AGV foi similar (média de 7.7 mas, no omaso, o valerato (90.0 foi mais metabolizado do que o butirato (59.6 propionato (69.8 e acetato (51.7 . A correlação entre o metabolismo de AGV e o índice mitótico foi positiva no rúmen e no omaso. Concluiu-se que o potencial de metabolismo e de absorção de AGV por unidade de área do omaso é mais alto do que o do rúmen. A variação da capacidade de absorção do rúmen e do omaso ocorre na mesma direção e existem indícios de que os fatores capazes de estimular a proliferação da parede do rúmen são também capazes de estimular a parede do omaso