Myopodin is an F-actin bundling protein with multiple independent actin-binding regions

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin’s ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

     

Sex determination in skeletal remains from the medieval Eastern Adriatic coast – discriminant function analysis of humeri

Aim

To investigate the usefulness of humerus measurement for sex determination in a sample of medieval skeletons from the Eastern Adriatic Coast. Additional aim was to compare the results with contemporary female population.

Methods

Five humerus measurements (maximum length, epicondylar width, maximum vertical diameter of the head, maximum and minimum diameter of the humerus at midshaft) for 80 male and 35 female medieval and 19 female contemporary humeri were recorded. Only sufficiently preserved skeletons and those with no obvious pathological or traumatic changes that could affect the measurements were included. For ten samples, analysis of DNA was performed in order to determine sex using amelogenin.

Results

The initial comparison of men and women indicated significant differences in all five measures (P < 0.001). Discriminant function for sex determination indicated that as much as 85% of cases could be properly categorized, with better results in men (86%) than women (80%). Furthermore, the comparison of the medieval and contemporary women did not show significant difference in any of the measured features. Sex results obtained by anthropological and DNA analysis matched in all 10 cases.

Conclusion

The results indicate that humerus measurement in Croatian medieval population may be sufficient to determine the sex of the skeleton. Furthermore, it seems that secular changes have not substantially affected contemporary population, suggesting that the results of this study are transferable to contemporary population as well.Once the skeletal remains are uncovered, anthropologists initially aim to reconstruct the biological profile of the person, which includes sex, age, and height estimation. During the reconstruction process, numerous issues may arise, including bone fragmentation and poor preservation of skeletal remains, coupled with the complexity of human skeleton (1,2). Sex determination is one of the first and basic steps of assessing the biological profile. Although the analysis of DNA is the most reliable method for sex determination (3), it is also the most expensive and time consuming method, which can also be hindered by local conditions. This may especially be true in cases of poor preservation of the remains, inhibitors effects, or a small amount of extracted DNA from the sample.In absence of DNA results, skeletal remains can be used to infer subject’s sex via two methods, morphological and anthropometric. The morphological approach is based on the examination of the bones that show the strongest sexual dimorphism, principally the skull and the pelvis (4). However, this method is not always reliable, especially if the skull is fragmented or incomplete. Age can also affect the results, especially in elderly women, in which morphological characteristics of the skull tend to resemble those of men (5). Although morphological methods are very important for a preliminary sex assessment, they additionally rely on the experience of the examiner and are therefore rather subjective and unreliable.The second approach is based on anthropometric analysis, which relies on the bone measurements. The main analytic approach is based on discriminant function analysis, which attempts to classify subjects into each of the sexes, by using either one or more bones (6). This kind of analysis is a very important quantitative method (7) for sex determination as it reduces the subjectivity of the examiner (2,4,8). So far, only a few such studies have been published using Croatian bone samples. These include medieval and contemporary femurs and tibias (8-11) and medieval and contemporary mandibles and teeth (6,12). Such studies are important, since clear differences were observed in different populations (13-15), making this a locally-specific feature that requires the development of regional standards, applicable for local population (16).Besides already used femurs and tibias, humerus is another long bone from the body that is presumably informative for sex determination. This idea was initially derived from the empirical investigations of the skeletal remains, and further supported by the previously reported sexual dimorphism of humeri (7,17), even in cases of severe bone fragmentation (18). Furthermore, such location-specific results may be of interest in modern forensics as well, since observed changes in the skeleton marked predominantly by the increase in height (19), appear to be proportional, with no indication of sexual dimorphism in ancient and modern samples (20). Therefore, the aim of this study was to investigate the possibility to determine the sex of the subject based on anthropometric analysis of humeri measures.     

Effects of a lifestyle-related risk factor modification intervention on lifestyle changes among patients with coronary artery disease in Nepal

ObjectiveTo investigate the effect of a lifestyle-related risk factor modification intervention on coronary artery disease (CAD) patients’ lifestyle changes.MethodA randomized controlled study was conducted in Nepal. A total of 224 CAD patients (112 in each study group) were included at baseline, and 196 patients (98 in each group) completed the one-month follow-up. Patients in the intervention group (IG) received nurse-led intervention in addition to the usual care. Face-to face and telephone interview was conducted using standard questionnaires to collect data on lifestyle-related risk factors; smoking, alcohol consumption, diet, body mass index, stress, adherence to medical therapy, and physical activity. General linear model repeated measure analysis was used to analyse the effects of the intervention.ResultsBased on self-reported data we found significant improvement in lifestyle-related risk factor habits in the IG compared with the usual care group with respect to diet (p < 0.001), physical activity (p < 0.001), medication adherence (p < 0.001) and stress (p < 0.001) at one-month follow-up.ConclusionLifestyle-related risk factor modification intervention can positively influence health risk habits, even when it is less intensive but supplemented with information leaflets.Practical implicationsNurse-led one-time intervention may successfully deliver counselling to improve healthy lifestyle among underserved CAD patients.     

De novo mTOR inhibitor‐based immunosuppression in ABO‐incompatible kidney transplantation

ABO‐incompatible (ABOi) kidney transplantation (KTx) has become an accepted therapeutic option in renal replacement therapy for patients without a blood group‐compatible living donor. Using different desensitization strategies, most centers apply B‐cell depletion with rituximab and maintenance immunosuppression (IS) with tacrolimus and mycophenolic acid. This high load of total IS leads to an increased rate of surgical complications and virus infections in ABOi patients. Our aim was to establish ABOi KTx using an immunosuppressive regimen, which is effective in preventing acute rejection without increasing the risk for viral infections. Therefore, we selected a de novo immunosuppressive protocol with low‐dose calcineurin inhibitor and the mTOR inhibitor everolimus for our ABOi program. Here, we report the first 25 patients with a complete three‐yr follow‐up treated with this regimen. Three‐yr patient survival and graft survival were 96% and 83%. The rate of acute T‐cell‐mediated rejections was low (12%). Cytomegalovirus (CMV) infection was evident in one patient only (4%). Surgical complications were common (40%), but mild in 80% of cases. We demonstrate that ABOi KTx with a de novo mTOR inhibitor‐based regimen is feasible without severe surgical or immunological complications and a low rate of viral infections.     

G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade

OBJECTIVES: In human end-stage heart failure as well as in experimental animal models of heart failure, G-protein-coupled receptor kinase activity (GRK) is increased while beta-adrenoceptor responsiveness is diminished. In animal studies, beta-adrenoceptor blockers reverse the GRK-mediated desensitization and down-regulation of myocardial beta-adrenoceptors. The aim of this study was to investigate whether alterations in GRK activity are an early or late accompaniment of human heart failure and whether also in humans beta-adrenoceptor blocker treatment is able to influence myocardial GRK activity. METHODS: We assessed in right atria, obtained from patients at different stages of heart failure, treated with or not treated with beta-adrenoceptor blockers, and in the four chambers of explanted hearts, obtained from patients with end-stage heart failure, beta-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and GRK activity (by an in vitro rhodopsin phosphorylation assay). RESULTS: With increasing severity of heart failure, plasma noradrenaline levels increased while myocardial beta-adrenoceptor density decreased with a maximum in GRK activity in end-stage heart failure. However, in relation to the progression of heart failure, we found that GRK activity transiently increased at an early stage of heart failure (NYHA I and II) but decreased back to control values in patients at NYHA III and IV. beta-Adrenoceptor blockers were able to reduce the early increase in GRK activity at NYHA I and II to control levels, whereas in those patients who did not have increased GRK activity (NYHA III and IV), they had only a marginal effect. CONCLUSION: According to our results, an increase in GRK activity is an early and transient event in the course of heart failure that can be prevented by beta-adrenoceptor blocker treatment.