Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein HY 322 Gene of Vibrio alginolyticus

In this study,Hy322 gene was cloned from Vibrio alginolyticus.The total length of its gene was 969 bp,and it could encode 322 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Hy322 of V.alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools.The results showed that Hy322 is an unstable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to α-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and V.harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.HY322 contains a FliN super family conserved domain associated with Flagellar motor switch.Bioinformatics analysis showed that the B-cell preponderant epitopes of Hy322 might be localized in the regions of 32-33,100-102,138-140,215-216,235-238 and 246-249.The 3D structure model of Hy322 subunit was simulated by SWISS-MODEL software and itwas found that the yscQ of Yersinia were similar and the similarity was 42.25%.In this study,the feasibility of Hy322 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.

Manganese-Catalyzed Regioselective Hydroboration of Quinolines via Metal-Ligand Cooperation

Selective reduction of readily available N-heteroarenes is important in both organic synthesis and chemical biology.Herein,we describe ligand-controlled regiodivergent hydroboration of quinolines using well-defined amido-manganese catalysts,with an emphasis on the rarely reported 1,4-regioselectivity.Mechanistic studies showed that 1,2-hydroboration of quinoline was kinetically favorable and reversible,whereas 1,4-hydroboration was under thermodynamic control.Using a 1-methyimidazolebased pincer amido-manganese complex as the catalyst,cooperative C-H…N andπ…πnoncovalent interactions between the 1-methyimidazole moiety and quinoline substrates enabled kinetic accessibility of 1,4-hydroboration,giving thermodynamically favored 1,4-hydroborated quinolines as the major products.On this basis,Mn-catalyzed 1,4-hydroboration of a series of substituted quinolines proceeded smoothly in high yields.A high turnover number of 2500 was achieved in this reaction with satisfying regioselectivity.This transformation could be further applied to the C3-selective functionalization of quinolines,highlighting the synthetic utility of this methodology.In contrast,using a pyridine-based pincer amido-manganese complex as the catalyst,which lacked the C-H…N interaction,the free-energy barrier for 1,4-hydroboration significantly increased through a N-B…N interaction between the“HMn-NB”species and quinoline,resulting in the kinetically favored 1,2-hydroboration product with excellent regioselectivity.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization

Auditory neuropathy spectrum disorder(ANSD)represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function,but with the preservation of outer hair cell function.ANSD represents up to 15%of individuals with hearing impairments.Through mutation screening,bioinformatic analysis and expression studies,we have previously identified several apoptosis-inducing factor(AIF)mitochondria-associated 1(AIFM1)variants in ANSD families and in some other sporadic cases.Here,to elucidate the pathogenic mechanisms underlying each AIFM1 variant,we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system and constructed AIF-wild type(WT)and AIF-mutant(mut)(p.T260A,p.R422W,and p.R451Q)stable transfection cell lines.We then analyzed AIF structure,coenzyme-binding affinity,apoptosis,and other aspects.Results revealed that these variants resulted in impaired dimerization,compromising AIF function.The reduction reaction of AIF variants had proceeded slower than that of AIF-WT.The average levels of AIF dimerization in AIF variant cells were only 34.5%-49.7%of that of AIF-WT cells,resulting in caspase-independent apoptosis.The average percentage of apoptotic cells in the variants was 12.3%-17.9%,which was significantly higher than that(6.9%-7.4%)in controls.However,nicotinamide adenine dinucleotide(NADH)treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells.Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD,and introduce NADH as a potential drug for ANSD treatment.Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.