Activation of Voltage-gated Calcium Current by Action Potentials and Modulation by G Proteins

Voltage-gated calcium channels are ubiquitously expressed in neurons and are of vital importance to proper cellular functioning. Calcium entry into cells via activation of voltage-gated calcium channels controls a wide variety of cellular functions including neurotransmitter release, muscle excitation-contraction coupling, gene regulation and activation of signaling cascades. Regulation of calcium channels via activation of G protein-coupled receptors is a prominent mechanism of calcium current inhibition and neurotransmitter release. An intriguing characteristic of this modulatory pathway is its voltage dependence whereby the degree of calcium current inhibition varies depending on the membrane voltage of the cell, and is susceptible to activity-dependent relief by trains of action potentials. Previously, it has been suggested that in chick ciliary ganglion neurons, somatostatin inhibits calcium current in a voltage dependent manner. Interestingly, the specific characteristics of the inhibition varied depending on the recording configuration used to collect data. Thus, I measured the voltage-dependence of somatostatin-mediated calcium current inhibition in individual ciliary ganglion neurons using the whole-cell and perforated patch configuration of voltage clamp recordings. The results indicate that the cytoplasmic dialysis that occurs during whole-cell recordings enhances the voltage dependence of calcium current inhibition and suggests that there is a greater concentration of activated G protein subunits in this configuration. While much is known about step depolarization-evoked calcium current and the kinetic changes that accompany G protein-mediated inhibition, relatively little is known about the effects of kinetic slowing on AP-evoked calcium current. Therefore, I used a modification of action potential waveforms was used to determine the effect of G protein activation on the kinetics of single action potential-evoked calcium current. The results demonstrate that kinetic slowing does not alter the time course of action potential-evoked calcium current and suggests that modulated channels may not contribute to AP-evoked calcium current

Regulation and Kinase Activity of the Trk Family of Receptor Tyrosine Kinases

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. Given the high homology between the Trks and their use of overlapping signaling pathways, the key question to be addressed is how the activation of the different Trks can lead to distinct cellular outcomes. To this end, I first sought to determine the mechanism of autoregulation for the Trk tyrosine kinase domain (TKD). The Trk TKDs are members of the insulin receptor kinase (IRK) superfamily and recent data suggest that the IRK family displays a wide array of autoinhibitory mechanisms. To determine where TrkA and the closely related Ror2 TKD (from an unconventional Wnt receptor) lie in this spectrum, we determined the crystal structures of the kinase domains of these RTKs. In both cases, the conformation of the activation loop resembles the IRK activation loop conformation, with subtle but notable differences in the case of Ror2. These findings aid in understanding the range of autoinhibitory mechanisms of the IRK family, in addition to providing a foundation for deciphering consequences of TKD mutations in this family. I also observed crystallographic dimers of the inactive TrkA TKD that resemble those seen for other RTK TKDs - which may aid in understanding the reported pre-formed inactive TrkA dimers observed in cells. To understand the molecular basis for differences in signaling specificity of the Trk receptors, I investigated whether the TrkA and TrkB TKDs differ in their intrinsic kinase activities. I show that the TrkA TKD autophosphorylates itself faster than its TrkB counterpart. However, this difference of autophosphorylation is not due to a difference in kinase activity per se. Rather, my data indicate that the difference in autophosphorylation may arise because of self-association of the TrkA TKD that does not occur with TrkB. My work sheds light on potential differences between TrkA and TrkB signaling, as well as providing a quantitative understanding of Trk TKD activation, which is useful for effective and selective inhibitor design

Implementasi Pariwisata Halal di Nusa Tenggara Barat (Studi Kasus Kawasan Ekonomi Khusus Mandalika, Kabupaten Lombok Tengah)

Peraturan daerah nomor 2 tahun 2016 Tentang Pariwisata halal adalah peraturan yang dibuat oleh Pemerintah Provinsi Nusa Tenggara Barat. Tujuan di bentuknya konsep pariwisata halal bagi pemerintah Provinsi Nusa Tenggara Barat adalah untuk meningkatkan nilai budaya dengan identitas islami yang dianggap sebagai wujub kearifan lokal. Kawasan Ekonomi Khusus (KEK) Mandalika adalah salah satu destinasi wisata yang sering dikunjungi oleh wisatawan lokal maupun wisatawan mancanegara. Akan tetapi di restoran atau hotel tersebut tidak hanya menyediakan makanan dan minuman halal, tetapi juga makanan dan minuman non halal. Sehingga fokus penelitian ini adalah untuk mengetahui bagaimana implementasi Pariwisata Halal di Kawasan Ekonomi Khusus (KEK) Mandalika, Kabupaten Lombok Tengah. Pendekatan yang peneliti gunakan dalam penelitian ini adalah pendekatan kualitatif. Teknik pengumpulan data yang digunakan yaitu wawancara, observasi, dan dokumentasi. Hasil yang peneliti dapatkan dari penelitian adalah Standar dari pariwisata halal masih bersifat rancu sehingga masih banyak orang yang belum mengerti tentang standar dari pariwisata halal itu sendiri. Hal tersebut dikarenakan kurangnya sosialisasi terhadap dinas-dinas atau organisasi yang terkait, sehingga mengakibatkan kesalahan persepsi terhadap konsep dari pariwisata halal itu sendiri. Kebijakan yang dijalankan oleh pemerintah provinsi atau dinas pariwisata provinsi belum dijalankan secara maksimal. Terbukti dari tim halal yang belum dibentuk oleh Dinas Pariwisata Provinsi. Komunikasi yang dijalankan oleh Dinas Pariwisata Provinsi kepada dinas-dinas dan organisasi yang terkait dengan pariwisata terbilang belum maksimal, hal tersebut dikarenakan Tim halal yang belum terbentuk. Tim halal bertugas untuk mensosialilasikan tentang pariwisata halal kepada dinas atau organisasi terkait agar pihak dari dinas pariwisata dari kabupaten dan organisasi yang berkaitan dengan pariwisata dapat paham dengan konsep dan tujuan dari pariwisata halal. Kondisi social di Nusa Tenggara Barat khususnya kabupaten Lombok Tengah siap dengan konsep pariwisata halal. Bahkan tanpa adanya perda nomor 2 tahun 2016 tentang pariwisata halal, daerah kuta mandalika sudah mumpuni untuk dikunjungi oleh wisatawan-wisatawan muslim tanpa perlu khawatir soal kenyamanan, tempat ibadah, serta makanan atau minuman halal

Beta 2 glycoprotein I Valine247Leucine polymorphism in patients with antiphospholipid syndrome

Aim: Beta 2 Glycoprotein I (β2-GP I) takes part in the pathogenesis of antiphospholipid syndrome (APS). Valine247Leucine (Val247Leu) gene polymorphism of β2-GP I might affect the binding/production of anti-β2-GP I antibodies. Multiple studies are showing different frequencies of this polymorphism in various ethnic backgrounds; we aimed to determine the frequency and clinical importance of Val247Leu gene polymorphism of β2-GP I in patients with APS and healthy. Methods: Eighty-three patients with APS [68 primary APS, 15 APS with systemic lupus erythematosus (SLE)] and 63 healthy individuals were included. Β2-GP I Val247Leu polymorphism was determined by quantitative real time polymerase chain reaction and melting curve analysis. The presence of anti-β2-GP I antibodies was detected by ELISA in the patient group. Results: Allele and genotype frequencies were similar between patients and healthy controls (p=0,307). V allele and VV genotype frequencies were significantly higher in primary APS patients with thrombocytopenia (p=0.040). There was no significant difference between β2-GP I Val247Leu gene polymorphism and the anti-β2-GP IgM and IgG antibody levels in the patient group (p=0.631 and p=0.077, respectively) Conclusion: This is the first study investigating the β2-GP I Val247Leu gene polymorphism in the Turkish population. The frequencies of Val247Leu gene polymorphism of β2-GP I were not different between patients with APS and healthy individuals in line with the other studies in Caucasian populations. Significantly high levels of V allele and VV genotype frequencies in primary APS patients could offer further insight to into the pathogenesis of thrombocytopenia in APS

Thrombotic risk assessment in antiphospholipid syndrome: do noncriteria antibodies contribute?

BACKGROUND/AIM: In this cross-sectional study, it was aimed to test the predictive value of noncriteria antiphospholipid antibodies (aPL) in addition to the global antiphospholipid syndrome score (GAPSS) in predicting vascular thrombosis (VT) in a cohort of patients with APS and aPL (+) systemic lupus erythematosus (SLE). MATERIAL AND METHODS: This study included 50 patients with primary APS, 68 with SLE/APS, and 52 with aPL (+) SLE who were classified according to VT as VT ± pregnancy morbidity (PM), PM only or aPL (+) SLE. Antiphospholipid serology consisting of lupus anticoagulant (LA), anticardiolipin (aCL) immunoglobulin G (IgG)/IgM/IgA, antibeta2 glycoprotein I (aβ2GPI) IgG/IgM/IgA, antiphosphatidylserine/prothrombin (aPS/PT) IgG/IgM and antidomain-I (aDI) IgG was determined for each patient. The GAPSS and adjusted GAPSS (aGAPSS) were calculated for each patient, as previously defined. Logistic regression analysis was carried out with thrombosis as the dependent variable and high GAPSS, aCL IgA, aβ2GPI IgA, and aDI IgG as independent variables. RESULTS: The mean GAPSS and aGAPSS of the study population were 11.6 ± 4.4 and 9.6 ± 3.8. Both the VT ± PM APS (n = 105) and PM only APS (n = 13) groups had significantly higher GAPSS and aGAPSS values compared to the aPL (+) SLE (n = 52) group. The patients with recurrent thrombosis had higher aGAPSS but not GAPSS than those with a single thrombotic event. The computed area under the receiver operating characteristic curve demonstrated that a GAPSS ≥13 and aGAPSS ≥10 had the best predictive values for thrombosis. Logistic regression analysis including a GAPSS ≥13, aCL IgA, aβ2GPI IgA, and aDI IgG showed that none of the factors other than a GAPSS ≥13 could predict thrombosis. CONCLUSION: Both the GAPSS and aGAPSS successfully predict the thrombotic risk in aPL (+) patients and aCL IgA, aβ2GPI IgA, and aDI IgG do not contribute to high a GAPSS or aGAPSS

Anti-factor Xa antibodies in patients with antiphospholipid syndrome and their effects upon coagulation assays

- Introduction: The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS). - Methods: Serum from patients with APS (n = 59), systemic lupus erythematosus (SLE; n = 106), other autoimmune rheumatic disease (ARD; n = 63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (± AT-III). - Results: Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P <0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P <0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P <0.05) and did not correlate with binding to AT-III. - Conclusion: APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy

Oncogenic RET Kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans

To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation

Alterations in common marmoset gut microbiome associated with duodenal strictures

Chronic gastrointestinal (GI) diseases are the most common diseases in captive common marmosets (Callithrix jacchus). Despite standardized housing, diet and husbandry, a recently described gastrointestinal syndrome characterized by duodenal ulcers and strictures was observed in a subset of marmosets sourced from the New England Primate Research Center. As changes in the gut microbiome have been associated with GI diseases, the gut microbiome of 52 healthy, non-stricture marmosets (153 samples) were compared to the gut microbiome of 21 captive marmosets diagnosed with a duodenal ulcer/stricture (57 samples). No significant changes were observed using alpha diversity metrics, and while the community structure was significantly different when comparing beta diversity between healthy and stricture cases, the results were inconclusive due to differences observed in the dispersion of both datasets. Differences in the abundance of individual taxa using ANCOM, as stricture-associated dysbiosis was characterized by Anaerobiospirillum loss and Clostridium perfringens increases. To identify microbial and serum biomarkers that could help classify stricture cases, we developed models using machine learning algorithms (random forest, classification and regression trees, support vector machines and k-nearest neighbors) to classify microbiome, serum chemistry or complete blood count (CBC) data. Random forest (RF) models were the most accurate models and correctly classified strictures using either 9 ASVs (amplicon sequence variants), 4 serum chemistry tests or 6 CBC tests. Based on the RF model and ANCOM results, C. perfringens was identified as a potential causative agent associated with the development of strictures. Clostridium perfringens was also isolated by microbiological culture in 4 of 9 duodenum samples from marmosets with histologically confirmed strictures. Due to the enrichment of C. perfringens in situ, we analyzed frozen duodenal tissues using both 16S microbiome profiling and RNAseq. Microbiome analysis of the duodenal tissues of 29 marmosets from the MIT colony confirmed an increased abundance of Clostridium in stricture cases. Comparison of the duodenal gene expression from stricture and non-stricture marmosets found enrichment of genes associated with intestinal absorption, and lipid metabolism, localization, and transport in stricture cases. Using machine learning, we identified increased abundance of C. perfringens, as a potential causative agent of GI disease and intestinal strictures in marmosets.National Institutes of Health/[T32 OD010978]/NIH/Estados UnidosNational Institutes of Health/[P30-ES002109]/NIH/Estados UnidosUniversidad de Costa Rica/[803-C1-163]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Domain I of beta 2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

Objectives This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI). Methods Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. Results Significant inhibition with DI was possible with average inhibition of 23% (N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition (p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone (p = 0.0001 and p = 0.001, n = 10). Conclusions Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients