Genetic association of KCNJ10 rs1130183 with seizure susceptibility and computational analysis of deleterious non-synonymous SNPs of KCNJ10 gene

Establishing genetic basis of Idiopathic generalized epilepsies (IGE) is challenging because of their complex inheritance pattern and genetic heterogeneity. Kir4.1 inwardly rectifying channel (KCNJ10) is one of the independent genes reported to be associated with seizure susceptibility. In the current study we have performed a comprehensive in silico analysis of genetic variants in KCNJ10gene at functional and structural level along with a case–control analysis for the association ofrs1130183 (R271C) polymorphism in Indian patients with IGE. Age and sex matched 108epileptic patients and normal healthy controls were examined. Genotyping of KCNJ10rs1130183variation was performed using PCR-RFLP method. The risk association was determined by using odds ratio and 95% confidence interval. Functional effects of non-synonymous SNPs (nsSNPs) in KCNJ10 gene were analyzed using SIFT PolyPhen-2, I-Mutant 2.0, PANTHER and FASTSNP. Subsequently, homology modeling of protein three dimensional (3D) structures was performed using Modeller tool (9.10v) and compared the native protein with mutant for assessment of structure and stability. SIFT, PolyPhen-2, I-Mutant 2.0 and PANTHER collectively showed rs1130183, rs1130182 and rs137853073 SNPs inKCNJ10 gene affect protein structure and function. There was a considerable variation in the Root Mean Square Deviation (RMSD) value between the native and mutant structure (1.17?). Association analysis indicate KCNJ10rs1130183 did not contribute to risk of seizure susceptibility in Indian patients with IGE (OR- 0.38; 95%CI, 0.07–2.05) and T allele frequency (0.02%) was in concordance with dbSNP reports. This study identifies potential SNPs that may contribute to seizure susceptibility and further studies with the selected SNPs in larger number of samples and their functional analysis is required for understanding the variants of KCNJ10with seizure susceptibility.     

Hyper-IgD and periodic fever syndrome: a new MVK mutation (p.R277G) associated with a severe phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM# 260920) is a rare recessively-inherited autoinflammatory condition caused by mutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Here we report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non-consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, whereas anakinra showed positive responses only at high doses. The p.R277G mutation here described is a novel missense MVK mutation, and it has been detected in this case with a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.     

Clinical and molecular genetic study of infantile-onset Pompe disease in Chinese patients: Identification of 6 novel mutations

Pompe disease is an autosomal recessive disorder and is caused by a deficiency in acid alpha-glucosidase (GAA). A broad range of studies have been performed on Pompe patients from different countries. However, the clinical course and molecular basis of the disease in Mainland China have not been well defined. In the present study, we examined a total of 18 Chinese children with infantile-onset Pompe disease to better understand the clinical and genetic features in this population. The median age at symptom onset was 3.6 months (range: 1.7–6.8 months) and 6.3 months at diagnosis (range: 2.5–9.3 months). All but 1 patient died at a median age of 8.2 months (range: 4.7–18.7 months). Molecular analysis revealed 20 different mutations, 6 of which are novel (c.1356delC, c.378G > A, c.1827C > G, c.859-2 A > T, c.1551 + 2T > G, and c.1465G > T). The most common mutation in the study was c.1935C > A, accounting for 25% (9/36 alleles) of the mutations. Our study provides the first comprehensive examination of the clinical course of infantile-onset Pompe disease and mutations of the GAA gene for patients in Mainland China. Our results confirm the high prevalence of the c.1935C > A mutation, previously reported for other populations, in Mainland Chinese patients with infantile-onset Pompe disease. Furthermore, six novel mutations in the GAA gene are reported for the first time.     

From bicycle chain ring shape to gear ratio: Algorithm and examples

A simple model of the bicycle drive system with a non-circular front chain ring is proposed and an algorithm is devised for calculation of the corresponding Gear Ratio As a Function Of Crank Angle (GRAFOCA). It is shown that the true effective radius of the chain ring is always the perpendicular distance between the crank axis and the line through the chain segment between the chain ring and the cog. It is illustrated that the true effective radius of the chain ring at any crank angle may differ substantially from the maximum vertical distance between the crank axis and the chain ring circumference that is used as a proxy for the effective chain ring radius in several studies; in particular, the crank angle at which the effective chain ring radius is maximal as predicted from the latter approach may deviate by as much as 0.30 rad from the true value. The algorithm proposed may help in designing chain rings that achieve the desired GRAFOCA.     

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.     

Efficient implementation of a filtered back-projection algorithm using a voxel-by-voxel approach

The large amount of image data necessary for high-resolution 3D reconstruction of macromolecular assemblies leads to significant increases in the computational time. One of the most time consuming operations is 3D density map reconstruction, and software optimization can greatly reduce the time required for any given structural study. The majority of algorithms proposed for improving the computational effectiveness of a 3D reconstruction are based on a ray-by-ray projection of each image into the reconstructed volume. In this paper, we propose a novel fast implementation of the "filtered back-projection" algorithm based on a voxel-by-voxel principle. Our version of this implementation has been exhaustively tested using both model and real data. We compared 3D reconstructions obtained by the new approach with results obtained by the filtered Back-Projections algorithm and the Fourier-Bessel algorithm commonly used for reconstructing icosahedral viruses. These computational experiments demonstrate the robustness, reliability, and efficiency of this approach.     

多需求鱼池定价问题

本文考虑了多需求下的鱼池定价问题．假设有m个鱼池可供投放鱼类种群,n个顾客需要使用鱼池,每个顾客有多个鱼类种群需要投放到至多m个鱼池中去．鱼池所有者根据顾客的需求和报价进行重新定价和最终分配,在满足部分顾客的全部需求下最大化其收益．文中考虑了两种定价机制-单一定价和按比例定价．并结合背包算法,在按比例定价情形下设计了具有对数竞争比的算法,最大化鱼池所有者的收益．     

Post-translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasite-containing vacuole is driven by its N-terminal domain

How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartments is still poorly understood. The dense granules of the intracellular protozoan Toxoplasma gondii constitute an unusual secretory pathway that allows soluble export of the GRA proteins which become membrane-associated within the parasite replicative vacuole. This process relies on both the segregation of the proteins routed to the dense granules from those destined to the parasite plasma membrane and on the sorting of the secreted GRA proteins to their proper final membranous system. Here, we provide evidence that the soluble trafficking of GRA6 to the dense granules relies on the N-terminal domain of the protein, which is sufficient to prevent GRA6 targeting to the parasite plasma membrane. We also show that the GRA6 N-terminal domain, possibly by interacting with negatively charged lipids, is fundamental for proper GRA6 association with the vacuolar membranous network of nanotubes. These results support our emerging model: sorting of transmembrane GRA proteins to the host cell vacuole is mainly driven by the dual role of their N-terminal hydrophilic domain and is compartmentally regulated.     

流式细胞仪分选人外周血T淋巴细胞的方法建立与评价

目的:利用流式细胞仪同时分离外人周血单个核细胞中T淋巴细胞并检测其分离纯度及存活率。方法:本文采用流式细胞仪同时分选人外周血CD4~+、CD8~+T淋巴细胞为例,推而广之,采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞,采用流式细胞仪同时分选CD4~+、CD8~+T淋巴细胞,分离细胞再通过流式细胞仪回测其分离纯度并通过台盼蓝染色检测分离细胞的存活率。结果:采用此方法能有效人外周血细胞CD4~+、CD8~+T淋巴细胞,分选前CD4~+淋巴细胞纯度为(50.5±11.5)%、CD8~+T淋巴细胞纯度为纯度为(15.4±7.1)%;分选后CD4~+T淋巴细胞纯度为(94.3±1.3)%、CD8~+T淋巴细胞纯度为(93.6±1.6)%;分选后CD4~+T淋巴细胞存活率为(95.3±1.8)%,CD8~+T淋巴细胞存活率为(94.8±1.5)%,细胞的形态完整。结论:采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞后利用流式细胞仪分选的方法能够高效、快速的分离人外周血CD4~+、CD8~+T淋巴细胞,且存活率高,为进一步研究其功能提供了保证。采用不同的荧光抗体标记其他淋巴细胞亚群,也能高效、快速的分离出细胞。