Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly

Temperature is one of the most important environmental factors that affect organisms,especially ectotherms,due to its effects on protein stability.Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial.Here,we report the amino acid substitutions of phosphoglucose isomerase(PGI)related to thermostability in the Glanville fritillary butterfly(Melitaea cinxia,Lepidoptera:Nymphalidae).The PGI encoded by the most common allele in M.cinxia in the Chinese population(G3-PGI),which is more thermal tolerant,is more stable under heat stress than that in the Finnish population(D1-PGI).There are 5 amino acid substitutions between G3-PGI and D1-PGI.Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q,M49T,and I64V may increase PGI thermostability.These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI.Through molecular dynamics simulations,it was found that the amino acid at site 421 is more stable in G3-PGI,confining the motion of theα-helix 420-441 and stabilizing the interaction between 2 PGI monomers.The strategy for hightemperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species(Boloria eunomia,Aglais urticae,Colias erate,and Polycaena lua)concurrent with M.cinxia in the Tianshan Mountains of China,i.e.,convergent evolution in butterflies.

A New Hybrid Model of Amino Acid Substitution for Protein Functional Classification

In this paper, a new hybrid model of amino acid substitution is developed and compared with the others in previous works. The results show that the new hybrid model can characterize the protein sequences very well by calculating Fisher weights, which can denote how much the variants contribute to the classification.

Allelic functional variation of FimH among Salmonella enterica subspecies

Salmonella enterica has a wide diversity,with numerous serovars belonging to six different subspecies with dynamic animal-host tropism.The FimH protein is the adhesin mediating binding to various cells,and slight amino acid discrepancy significantly affects the adherence capacities.To date,the general function of FimH variability across dif-ferent subspecies of Salmonella enterica has not been addressed.To investigate the biological functions of FimH among the six Salmonella enterica subspecies,the present study performed several assays to determine biofilm for-mation,Caenorhabditis elegans killing,and intestinal porcine enterocyte cell IPEC-J2 adhesion by using various FimH allele mutants.In general,allelic mutations in both the lectin and pilin domains of FimH could cause changes in bind-ing affnity,such as the N79S mutation.We also observed that the N79S variation in Salmonella Dublin increased the adhesive ability of IPEC-J2 cells.Moreover,a new amino acid substitution,T260M,within the pilin domain in one subspecies llb strain beneficial to binding to cells was highlighted in this study,even though the biofilm-forming and Caenorhabditis elegans-killing abilities exhibited no significant differences in variants.Combined with point muta-tions being a natural tendency due to positive selection in harsh environments,we speculate that allelic variation T26oM probably contributes to pathoadaptive evolution in Salmonella enterica subspecies llb.

New real-time-PCR method to identify single point mutations in hepatitis C virus

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

4-Hydroxybenzyl-substituted amino acid derivatives from Gastrodia elata

Seven new 4-hydroxyben l-substituted amino acid derivatives (1-7), together with 11 known compounds, were isolated from an aqueous extract of the rhizomes of Go,arodia data Blume. Their structures were determined by spectroscopic and chemical methods. Compounds 1-3 are pyroglutamate derivatives containing 4-hydroxybenzyl units at the N atom and 4-7 are the first examples of natural products with the 4-hydroxybenzyl unit linked vici a thioether bond to 2-hydroxy-3mercaptopropanoic acid (4-6) and 2-hydroxy-4-mercaptobutanoic acid (7), which would be biogenetically derived from cysteine and homocysteine, respectively. The structures of 1 and 2 were verified hi synthesis, while the absolute configurations of 4, 5 and 7 were assigned using Mosher 's method based on the MPA determination rule of A5Rs values. The known compound 4-(hydroxymethy0-5-nifrobenzene1,2-diol (8) exhibited activity against Fe2 r--cysteine induced rat liver microsomal lipid peroxidation with 1050 values of 9.99 x 10 6 mon. (C) 2015 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.'s/. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Conservation and variability of hepatitis B core at different chronic hepatitis stages

BACKGROUND Since it is currently not possible to eradicate hepatitis B virus(HBV)infection with existing treatments,research continues to uncover new therapeutic strategies.HBV core protein,encoded by the HBV core gene(HBC),intervenes in both structural and functional processes,and is a key protein in the HBV life cycle.For this reason,both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies.Moreover,alterations in the protein sequence could serve as potential markers of disease progression.AIM To detect,by next-generation sequencing,HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies.METHODS Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage[chronic hepatitis B infection without liver damage(CHB,n=16),liver cirrhosis(LC,n=5),and hepatocellular carcinoma(HCC,n=17)].HBV DNA was extracted from patients’plasma.A region between nucleotide(nt)1863 and 2483,which includes HBC,was amplified and analyzed by next-generation sequencing(Illumina Mi Seq platform).Sequences were genotyped by distance-based discriminant analysis.General and intergroup nt and amino acid(aa)conservation was determined by sliding window analysis.The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences.RESULTS Three nt(nt 1900-1929,2249-2284,2364-2398)and 2 aa(aa 117-120,159-167)hyper-conserved regions were shared by all the clinical groups.All groups showed a similar pattern of conservation,except for five nt regions(nt 1946-1992,2060-2095,2145-2175,2230-2250,2270-2293)and one aa region(aa 140-160),where CHB and LC,respectively,were less conserved(P<0.05).Some group-specific conserved regions were also observed at both nt(2306-2334 in CHB and 1935-1976 and 2402-2435 in LC)and aa(between aa 98-103 in CHB and 28-30 and 51-54 in LC)levels.No differences in insertion and deletions frequencies were observed.An aa substitution(P79 Q)was observed in the HCC group with a median(interquartile range)frequency of 15.82(0-78.88)vs 0(0-0)in the other groups(P<0.05 vs CHB group).CONCLUSION The differentially conserved HBC and HBV core protein regions and the P79 Q substitution could be involved in disease progression.The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies.

Functional analysis of three amino acid residues of purR repressor, Trp147, Gln-218 and Gln-292 in Salmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DMA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G721(→A), C933(→T) and C1155(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.

Study of isolation of fluoroquinolone-resistant Ureaplasma urealyticum and identification of mutant sites

OBJECTIVE: To study the resistance mechanism of clinical isolates of Ureaplasma urealyticum resistant to fluoroquinolones. METHODS: Thirteen isolates of Ureaplasma urealyticum resistant to six fluoroquinolones were selected out of 184 clinical isolates and their QRDRs (quinolone resistance-determining region) gyrA, gyrB, parC and parE were amplified by PCR. Sequencing results were compared to those susceptible reference strains and a comparison of deduced amino acid sequences were performed. RESULTS: Sequence comparison revealed a C to A change at 87nt of gyrA QRDR leading to the substitution of Asp95 with glutamic acid and a C to T change at 50nt of parC QRDR leading to the substitution of Ser80 with leucine. CONCLUSION: These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with fluoroquinolone resistance of Ureaplasma urealyticum.

Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

The Hantaan virus(HTNV)and Seoul virus(SEOV)mutants have accumulated over time.It is important to determine whether their neutralizing epitopes have evolved,thereby making the current vaccine powerless.However,it is impossible to determine by using traditional plaque reduction neutralization test(PRNT),because it requires large numbers of live mutant strains.Pseudovirus-based neutralization assays(PBNA)were developed by employing vesicular stomatitis virus(VSV)backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG).56 and 51 single amino acid substitutions of glycoprotein(GP)in HTNV and SEOV were selected and introduced into the reference plasmid.Then the mutant pseudoviruses were generated and tested by PBNA.The PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG.53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully generated.Importantly,by using PBNA,we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants respectively.The novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and SEOV.And the current vaccine remains to be effective for the naturally occurring mutants.