Deletion of N-terminal rapsyn domains disrupts clustering and has dominant negative effects on clustering of full-length rapsyn

The peripheral muscle membrane protein rapsyn is essential for the formation and maintenance of high density acetylcholine receptor aggregates at the neuromuscular synapse. Rapsyn is concentrated at synaptic sites and is colocalized with acetylcholine receptors from the earliest stages of synaptogenesis. Previous studies have shown that recombinant rapsyn expressed in heterologous cells forms clusters, and acetylcholine receptors coexpressed with rapsyn are colocalized with rapsyn clusters. However, the molecular interactions involved in clustering of rapsyn are not well defined. To analyze the process of cluster formation by rapsyn we examined the formation of rapsyn clusters and complexes using mutant constructs specifically deleted for individual domains of rapsyn in the presence and absence of tagged, full-length rapsyn. Specific deletions of the tetratricopeptide repeat (TPR) domains 1 and 3 of rapsyn abrogated not only clustering of mutant rapsyns, but also, in a dominant negative fashion, the clustering of tagged, full-length rapsyn. We also analyzed rapsyn protein complexes isolated from cells transfected with tagged and untagged rapsyn. Our results show that both tagged and untagged rapsyn are present in immunoprecipitates of rapsyn from cotransfected cells, demonstrating that rapsyn molecules interact directly or indirectly to form oligomers. Mutants that were dominant negatives were also present in complexes containing tagged, full-length rapsyn. Together these results indicate that rapsyn forms clusters at the synapse by oligomerization, and suggest models for the mechanistic bases of this oligomerization via interactions mediated by TPRs 1 and 3.     

Lack of vitamin D receptor causes stress-induced premature senescence in vascular smooth muscle cells through enhanced local angiotensin-II signals

Objectives

The inhibition of the renal renin-angiotensin system by the active form of vitamin D contributes to the cardiovascular health benefits of a normal vitamin D status. Local production of angiotensin-II in the vascular wall is a potent mediator of oxidative stress, prompting premature senescence. Herein, our objective was to examine the impact of defective vitamin D signalling on local angiotensin-II levels and arterial health.

Methods

Primary cultures of aortic vascular smooth muscle cells (VSMC) from wild-type and vitamin D receptor-knockout (VDRKO) mice were used for the assessment of cell growth, angiotensin-II and superoxide anion production and expression levels of cathepsin D, angiotensin-II type 1 receptor and p57^Kip2. The in vitro findings were confirmed histologically in aortas from wild-type and VDRKO mice.

Results

VSMC from VDRKO mice produced more angiotensin-II in culture, and elicited higher levels of cathepsin D, an enzyme with renin-like activity, and angiotensin-II type 1 receptor, than wild-type mice. Accordingly, VDRKO VSMC showed higher intracellular superoxide anion production, which could be suppressed by cathepsin D, angiotensin-II type 1 receptor or NADPH oxidase antagonists. VDRKO cells presented higher levels of p57^Kip2, impaired proliferation and premature senescence, all of them blunted upon inhibition of angiotensin-II signalling. In vivo studies confirmed higher levels of cathepsin D, angiotensin-II type 1 receptor and p57^Kip2 in aortas from VDRKO mice.

Conclusion

The beneficial effects of active vitamin D in vascular health could be a result of the attenuation of local production of angiotensin-II and downstream free radicals, thus preventing the premature senescence of VSMC.     

Influence of estradiol treatment on bone marrow cell differentiation in collagenase-induced arthritis

Objective and design

Estrogen is one of the important regulators of the balance between bone formation and bone resorption that can modulate the levels and activity of certain growth factors and cytokines. In this study, we investigated the effect of 17β-estradiol (ED) on bone marrow (BM) cell differentiation in vivo and ex vivo in a mouse model of collagenase-induced osteoarthritis (CIOA).

Subject

ICR (CD-2) female mice were used in present experiments (total number?=?75) and bone marrow cells were used for in vitro studies.

Treatment

Mice were orally fed under different schemes with 17β-estradiol at a dose of 2 μg or 4 μg for 30 days.

Methods

The effect of estradiol was estimated by histopathological, flow cytometry, and ELISA assays. Statistical differences were determined by one-way ANOVA.

Results

Estradiol treatment ameliorated cartilage destruction and osteophyte formation if started from day 0 of CIOA induction, attended with a decrease of uterine and ovarian weights. Long time treatment lowered the percentage of megakaryocyte/platelet (CD62P+) populations and osteoclast (RANK+) populations in BM. Cells obtained from estradiol-treated CIOA mice showed inhibited capacity to differentiate into RANK+ and mesenchymal cells under osteoclastogenic conditions in vitro. Estrogen decreased serum IL-6 levels.

Conclusion

Results indicate a potential protective role for estrogen against the development of OA.

     

Investigating the temporal dynamics of electroencephalogram (EEG) microstates using recurrent neural networks

Electroencephalogram (EEG) microstates that represent quasi‐stable, global neuronal activity are considered as the building blocks of brain dynamics. Therefore, the analysis of microstate sequences is a promising approach to understand fast brain dynamics that underlie various mental processes. Recent studies suggest that EEG microstate sequences are non‐Markovian and nonstationary, highlighting the importance of the sequential flow of information between different brain states. These findings inspired us to model these sequences using Recurrent Neural Networks (RNNs) consisting of long‐short‐term‐memory (LSTM) units to capture the complex temporal dependencies. Using an LSTM‐based auto encoder framework and different encoding schemes, we modeled the microstate sequences at multiple time scales (200–2,000 ms) aiming to capture stably recurring microstate patterns within and across subjects. We show that RNNs can learn underlying microstate patterns with high accuracy and that the microstate trajectories are subject invariant at shorter time scales (≤400 ms) and reproducible across sessions. Significant drop in the reconstruction accuracy was observed for longer sequence lengths of 2,000 ms. These findings indirectly corroborate earlier studies which indicated that EEG microstate sequences exhibit long‐range dependencies with finite memory content. Furthermore, we find that the latent representations learned by the RNNs are sensitive to external stimulation such as stress while the conventional univariate microstate measures (e.g., occurrence, mean duration, etc.) fail to capture such changes in brain dynamics. While RNNs cannot be configured to identify the specific discriminating patterns, they have the potential for learning the underlying temporal dynamics and are sensitive to sequence aberrations characterized by changes in metal processes. Empowered with the macroscopic understanding of the temporal dynamics that extends beyond short‐term interactions, RNNs offer a reliable alternative for exploring system level brain dynamics using EEG microstate sequences.     

Decreased measles virus-neutralizing activity in sera from otosclerotic patients

HYPOTHESIS: Any kind of depressed systemic anti-measles reaction can lead to the induction of a local immune response in the inner ear and possibly to reactivation of bone turnover in this region. METHODS: Different dilutions of sera were tested for neutralizing activity against a constant viral concentration. The ability of measles virus to infect and replicate in the cell monolayer was detected by enumeration of living and growing cells with a colored reaction. RESULTS: Virus-neutralizing activity in the sera of patients with confirmed otosclerosis was significantly weaker than in that of healthy controls. When age- and sex-matched pairs were compared, the neutralizing activity in the healthy counterpart was higher in 5 cases. Nearly complete viral neutralization was achieved with samples containing inactivated complement and in IgG-containing fractions, but not in immunoglobulin-depleted samples. CONCLUSION: The present study is consistent with measles virus participation at least in the initiation of some cases of otosclerosis.     

Differentiation of obese patients at moderate or higher Findrisc score based on their atherogenic index

Objectives: The purpose of this study was to reveal different subgroups of patients with at least moderate risk of developing diabetes in the next 10 years, based on clustering of cardiovascular risk factors.

Methods: We performed a one-center cross-sectional study of adult patients (n = 109, median age 45 years) with Findrisc score of above 11 out of 26 maximum. We included in the cluster analysis anthropometrics, lipid and carbohydrate parameters obtained in oral glucose tolerance test (OGTT), insulin, C-peptide, creatinine, C-reactive protein, liver enzymes, beta-cell function, insulin sensitivity and insulin resistance (HOMA calculations). We also evaluated the atherogenic index of plasma (AIP).

Results: We identified three metabolic phenotypes of patients with at least moderate Findrisc score—one ‘male’ (cluster AM, n = 24), and two ‘female’ phenotypes (cluster AW, n = 9 and cluster BW, n = 76). Men were almost homogenous for their metabolic phenotype, with lower fat percentage than women (p < .05). Most of the women (cluster BW, n = 76) presented with better metabolic pattern i.e. lower insulin resistance, lower C-reactive protein, lower degree of obesity and visceral fat rating (p < .05), despite the higher fat percentage (p < .05). Some of the women, however, (cluster AW, n = 9) presented with parameters very similar to that of men (cluster AM) and significantly higher than in cluster BW. Despite the lack of significant differences in lipid parameters among clusters, AIP was significantly lower in cluster BW (p < .05).

Conclusion: Most of the women presented with clearly less unfavorable atherogenic risk than men. Two different phenotypes of obese women with at least moderate Findrisc score were revealed, and the level of inflammation seems to be the main discriminant factor. Larger prospective studies are required to elucidate whether those are really two different pathogenically phenotypes or if they belong to the same phenotype’s continuum.