An MLPA-based approach for high-resolution genotyping of disease-related multi-allelic CNVs

Copy number variation has recently been recognized as an important type of genetic variation that modifies human phenotypes. Copy number variants (CNVs) are being increasingly associated with various human phenotypes and diseases. However, the lack of an appropriate method that allows fast, inexpensive and, most importantly, accurate CNVs genotyping significantly hampers CNV analysis. This limitation especially affects the analysis of multi-allelic CNVs that frequently modify various phenotypes. Recently, we developed a multiplex ligation-dependent probe amplification (MLPA)-based strategy for multiplex copy number genotyping and the validation of candidate CNV-miRNAs. Here we present the adaptation and optimization of this recently developed method for high-resolution genotyping of individual disease-related multi-allelic CNVs. We developed appropriate assays for three well-known and extensively studied CNVs: CNV-CCL3L1, CNV-DEFB, and CNV-UGT2B17, which have been associated with various human phenotypes including inflammation-related and infectious diseases. With the use of these assays we identified several general factors that allow to increase the resolution of the copy number genotyping. Performed experiments confirmed the high reproducibility and accuracy of the obtained genotyping results. The reliability of the results and relatively low per-genotype cost makes this strategy an attractive method for large-scale experiments such as genotype–phenotype association studies.     

Synthesis, structure, and heterogeneous catalytic activity of tungsten chalcogenide cubane cluster containing alkaline earth metal complex

A new compound containing a cubane tungsten chalcogenide cluster [W₄(μ₃-Te)₄(CN)₁₂]⁶⁻ and Ca²⁺ complex units has been prepared by the reaction of aqueous solution of K₆[W₄(μ₃-Te)₄(CN)₁₂] · 5H₂O with the solution of a Ca(NO₃)₂ and phen(1,10-phenanthroline) (1:2 molar ratio) in a solvent mixture of H₂O/EtOH. The structure of [{Ca(phen)₂(H₂O)}{Ca(phen)(H₂O)₄}{Ca(phen)₂(H₂O)₃}][W₄Te₄(CN)₁₂] · 5H₂O 1 has been determined by X-ray crystallography. Compound 1 contains [{Ca(phen)(H₂O)₄}{Ca(phen)₂(H₂O)₃}][W₄(μ₃- Te)₄(CN)₁₂] units bridged by {Ca(phen)₂(H₂O)}²⁺ units to form an one-dimensional zigzag chain structure. Interestingly, compound 1 showed a heterogeneous catalytic activity in the transesterification of a range of esters with methanol under the mild conditions. Moreover, it can be reused without any loss of activity through 10 runs with ester.     

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K_5CN05 > 200) and yield (Y_5CN05 > 99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K_5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.     

A new approach for producing polyclonal antibodies using impure antigens

A new convenient approach has been designed to produce polyclonal antibodies (PcAb). The approach is based on the principle of the immunoglobulin (Ig) class switch in the immune response. We produced six different antibodies (Ab) against calcineurin A subunit (CNA). CNA, His-tagged calcineurin A subunit (His-CNA), single chain calcineurin (CNB-CNA) and single chain calcineurin-calmodulin complex (CaM-CNB-CNA) were expressed in Escherichia coli (E. coli) BL21 strain, and they were used to immunize male BALB/c mice. These Ab were examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The results clearly demonstrated that the specificity of the Ab produced by different protein samples was much higher than that of the Ab produced by a single sample. We used CNA, CaM-CNB-CNA and His-CNA to immunize mice in turn and obtained monospecific PcAb against CNA fortunately by our new approach. Remarkably, our approach not only offered a simple and general alternative to other methods for producing PcAb described previously, but also disclosed a novel process of immunization that could be used to produce monoclonal antibodies (mAb).     

HIV-1 CN54 Pol P51抗原高效表达、纯化、复性及在抗体检测中的应用

为获得可溶性高纯度HIV-1中国株CN54PolP51抗原,将携带CN54polp51基因的重组质粒pTHioHisA51转化大肠杆菌BL21codonplus-RIL,用IPTG进行诱导表达。用Chelating SepharoseFF-Ni亲和层析柱及DEAE Sepharose Fast Flow阴离子交换层析柱纯化目的蛋白,采用透析复性法得到可溶性抗原,Western blotting检测目的蛋白。用纯化的P51抗原蛋白标记辣根过氧化物酶及包被酶标板进行双抗原夹心法ELISA检测。结果显示P51以包涵体的形式表达,表达量占菌体总蛋白的50%,经两步层析和透析复性,目的蛋白纯度大于95%。Western blotting和双抗原夹心法ELISA检测均显示了良好的灵敏度和特异性。本研究可以为HIV-1疫苗研究和开发检测试剂提供支持。     

UDP-glucuronosyltransferase 1A1 (UGT1A1) gene haplotypes and their effect on serum bilirubin concentration in healthy Indian adults

The aim of the present study was to investigate the allele and genotype frequencies and haplotype structures of the variants in the UGT1A1 gene and their association with serum bilirubin levels in healthy adults. Total serum bilirubin levels were measured in 300 healthy adults (normal hematology and liver function test) and genotyping of seven SNPs was performed by PCR-RFLP, Gene Scan analysis and direct sequencing on the ABI Prism 310 Genetic Analyzer. Of the seven SNPs, four were found to be polymorphic and the frequencies of minor alleles were 0.336, 0.431, 0.353 and 0.066 for − 53(TA)7, − 3279G, − 3156A and 211A respectively. Individuals who carried the − 53(TA)7, − 3279G and − 3156A mutant alleles in homozygous or heterozygous states had significantly higher mean serum bilirubin levels. Five major promoter haplotypes were observed: − 53(TA)6/− 3279T/− 3156G was the most common haplotype, followed by − 53(TA)7/− 3279G/− 3156A, − 53(TA)6/− 3279G/− 3156G, − 53(TA)6/− 3279G/− 3156A and − 53(TA)7/− 3279T/− 3156G with an estimated frequency of 0.445, 0.230, 0.083, 0.065 and 0.050 respectively. Furthermore, the mutant haplotype (− 53(TA)7/− 3279G/− 3156A) was found to have a significant effect on bilirubin concentrations. Promoter polymorphisms and a common haplotype of the UGT1A1 gene are associated with serum bilirubin concentrations and could be a genetic risk factor for hyperbilirubinemia in Indians.     

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations

Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the ‘CN network’ hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the ‘CN network’ hypothesis has been revisited and the present study suggested that ‘CN network’ should be accounted in terms of ‘structural contacts’ and ‘structural strengths’ in order to precisely describe order of structural stabilities of TFTs.