Non-synonymous Amino Acid Substitutions Research Articles

Adaptive changes in BMAL2 with increased locomotion associated with the evolution of unihemispheric slow-wave sleep in mammals.

Marine mammals, especially cetaceans, have evolved a very special form of sleep characterized by unihemispheric slow-wave sleep (USWS) and a negligible amount or complete absence of rapid-eye-movement sleep; however, the underlying genetic mechanisms remain unclear. Here, we detected unique, significant selection signatures in basic helix-loop-helix ARNT like 2 (BMAL2; also called ARNTL2), a key circadian regulator, in marine mammal lineages, and identified two nonsynonymous amino acid substitutions (K204E and K346Q) in the important PER-ARNT-SIM domain of cetacean BMAL2 via sequence comparison with other mammals. In vitro assays revealed that these cetacean-specific mutations specifically enhanced the response to E-box-like enhancer and consequently promoted the transcriptional activation of PER2, which is closely linked to sleep regulation. The increased PER2 expression, which was further confirmed both in vitro and in vivo, is beneficial for allowing cetaceans to maintain continuous movement and alertness during sleep. Concordantly, the locomotor activities of zebrafish overexpressing the cetacean-specific mutant bmal2 were significantly higher than the zebrafish overexpressing the wild-type gene. Subsequently, transcriptome analyses revealed that cetacean-specific mutations caused the upregulation of arousal-related genes and the downregulation of several sleep-promoting genes, which is consistent with the need to maintain hemispheric arousal during USWS. Our findings suggest a potential close relationship between adaptive changes in BMAL2 and the remarkable adaptation of USWS and may provide novel insights into the genetic basis of the evolution of animal sleep.

Association between the FAAH C385A variant (rs324420) and obesity-related traits: a systematic review.

Overweight and obesity are the consequence of a sustained positive energy balance. Twin studies show high heritability rates pointing to genetics as one of the principal risk factors. By 2022, genomic studies led to the identification of almost 300 obesity-associated variants that could help to fill the gap of the high heritability rates. The endocannabinoid system is a critical regulator of metabolism for its effects on the central nervous system and peripheral tissues. Fatty acid amide hydrolase (FAAH) is a key enzyme in the inactivation of one of the two endocannabinoids, anandamide, and of its congeners. The rs324420 variant within the FAAH gene is a nucleotide missense change at position 385 from cytosine to adenine, resulting in a non-synonymous amino acid substitution from proline to threonine in the FAAH enzyme. This change increases sensitivity to proteolytic degradation, leading to reduced FAAH levels and increased levels of anandamide, associated with obesity-related traits. However, association studies of this variant with metabolic parameters have found conflicting results. This work aims to perform a systematic review of the existing literature on the association of the rs324420 variant in the FAAH gene with obesity and its related traits. A literature search was conducted in PubMed, Web of Science, and Scopus. A total of 645 eligible studies were identified for the review. After the identification, duplicate elimination, title and abstract screening, and full-text evaluation, 28 studies were included, involving 28 183 individuals. We show some evidence of associations between the presence of the variant allele and higher body mass index, waist circumference, fat mass, and waist-to-hip ratio levels and alterations in glucose and lipid homeostasis. However, this evidence should be taken with caution, as many included studies did not report a significant difference between genotypes. These discordant results could be explained mainly by the pleiotropy of the endocannabinoid system, the increase of other anandamide-like mediators metabolized by FAAH, and the influence of gene-environment interactions. More research is necessary to study the endocannabinoidomic profiles and their association with metabolic diseases.

Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector.

Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Molecular characterization of complete coding sequence of the MBL1 gene in the Indian Buffalo (Bubalus bubalis) breed

Mannose-binding lectin (MBL) is a member of the collectin protein family that binds a broad range of microorganisms and activates the lectin-complement pathway of innate immunity. A number of mutations have been found in both the coding as well as the non-coding regions of the MBL1 gene in various species, of which several variations affected the assembly of MBL1, thus leading to a low level of plasmic MBL and innate immune dysfunctions. In the present study, we have reported molecular cloning and characterization of the complete coding sequence of the MBL1 gene in the Indian buffalo breed Murrah. A 951 bp fragment of the MBL1 gene was amplified, cloned and sequenced. Multiple sequence alignment with other buffalo and cattle breeds revealed that the Murrah buffalo MBL1 CDS was 98.1-99.6% homologous to other buffalo breeds, and 98.3-98.5% similar to cattle breeds at nucleotide level. It was 96.8-98.8% homologous to buffalo breeds and 96.8-97.2% similar to cattle breeds at amino acid level. The amino acid sequence of the Murrah buffalo MBL1 contained two non-synonymous amino acid substitutions (L204P and S180P). Further, PCR-RFLP was performed to screen 50 Murrah buffalo for the presence of SNPs, g.855G>A in intron I and g.2686T>C, as well as g.2651G>A in exon 2 region of the MBL1 gene. The ApaI/intron I PCR-RFLP assay revealed a polymorphic pattern with three genotypes viz., AG (90%), GG (8%) and AA (2%), with allelic frequencies 0.94 for G and 0.06 for A. HaeIII/exon 2 PCR-RFLP assay exhibited the presence of three genotypes, namely, TC (66%), TT (32%) and CC (2%) with allelic frequencies 0.15 for T and 0.85 for C. StyI/exon 2. PCR-RFLP assay showed a monomorphic pattern for g.2651G>A with GG genotype only. We further examined the association of these SNPs with milk production traits and somatic cell score (SCS), and found no significant difference for any of the traits. Since the present study has formulated the results on the basis of a relatively small sample size, further studies with a larger sample size are required to validate the effects of polymorphisms.

Development of a Single-nucleotide Polymorphism Genotyping Assay for the Rapid Detection of Vancomycin-intermediate Resistance in Staphylococcus aureus Epidemic Lineage ST5.

Vancomycin is a treatment option for patients with severe methicillin-resistant Staphylococcus aureus (MRSA) infection. Unfortunately, reduced susceptibility to vancomycin in S. aureus is becoming increasingly common. We developed a method for the rapid and accurate diagnosis of vancomycin-intermediate resistant S. aureus (VISA). We performed a microbial genome-wide association study to discriminate between VISA and vancomycin-susceptible S. aureus (VSSA) using 42 whole-genome sequences. A TaqMan single-nucleotide polymorphism (SNP) genotyping assay was developed to detect target SNPs in VISA strains. Four SNPs in the VISA strains resulting in nonsynonymous amino-acid substitutions were associated with reduced susceptibility to vancomycin: SA_RS01235 (G203S), SA_RS09725 (V171A), SA_RS12250 (I48F), and SA_RS12550 (G478A). These four SNPs were mainly detected in the typical hospital-associated sequence type (ST)5 clonal lineage. The TaqMan assay successfully detected all four SNPs using as little as 0.2 ng DNA per reaction. Using 10 VSSA and VISA clinical strains each, we validated that the assay accurately discriminates between VISA and VSSA. The TaqMan SNP genotyping assay targeting four SNPs may be an alternative to current standard methods for the rapid detection of vancomycin-intermediate resistance in S. aureus epidemic lineage ST5.

A Novel CMY Variant Confers Transferable High-Level Resistance to Ceftazidime-Avibactam in Multidrug-Resistant Escherichia coli.

Here, our objective was to explore the molecular mechanism underlying ceftazidime-avibactam resistance in a novel CMY-178 variant produced by the clinical Escherichia coli strain AR13438. The antibiotic susceptibility of the clinical isolate, its transconjugants, and its transformants harboring transferable blaCMY were determined by the agar dilution method. S1-PFGE, cloning experiments, and whole-genome sequencing (WGS) were performed to investigate the molecular characteristics of ceftazidime-avibactam resistance genes. Kinetic parameters were compared among the purified CMY variants. Structural modeling and molecular docking were performed to assess the affinity between the CMYs and drugs. The horizontal transferability of the plasmid was evaluated by a conjugation experiment. The fitness cost of the plasmid was analyzed by determining the maximal growth rate, the maximum optical density at 600 nm (OD600), and the duration of the lag phase. AR13438, a sequence type 457 E. coli strain, was resistant to multiple cephalosporins, piperacillin-tazobactam, and ceftazidime-avibactam at high levels and was susceptible to carbapenems. WGS and cloning experiments indicated that a novel CMY gene, blaCMY-178, was responsible for ceftazidime-avibactam resistance. Compared with the closely related CMY-172, CMY-178 had a nonsynonymous amino acid substitution at position 70 (Asn70Thr). CMY-178 increased the MICs of multiple cephalosporins and ceftazidime-avibactam compared with CMY-172. The kinetic constant Ki values of CMY-172 and CMY-178 against tazobactam were 2.12 ± 0.34 and 2.49 ± 0.51 μM, respectively. Structural modeling and molecular docking indicated a narrowing of the CMY-178 ligand-binding pocket and its entrance and a stronger positive charge at the pocket entrance compared with those observed with CMY-172. blaCMY-178 was located in a 96.9-kb IncI1-type plasmid, designated pAR13438_2, which exhibited high transfer frequency without a significant fitness cost. In conclusion, CMY-178 is a novel CMY variant that mediates high-level resistance to ceftazidime-avibactam by enhancing the ability to hydrolyze ceftazidime and reducing the affinity for avibactam. Notably, blaCMY-178 could be transferred horizontally at high frequency without fitness costs. IMPORTANCE Ceftazidime-avibactam is a novel β-lactam-β-lactamase inhibitor (BLBLI) combination with powerful activity against Enterobacterales isolates producing AmpC, such as CMY-like cephalosporinase. However, in recent years, CMY variants have been reported to confer ceftazidime-avibactam resistance. We reported a novel CMY variant, CMY-178, that confers high-level ceftazidime-avibactam resistance with potent transferability. Therefore, this resistance gene is a tremendous potential menace to public health and needs attention of clinicians.

XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.

Weighted Single-Step GWAS Identifies Genes Influencing Fillet Color in Rainbow Trout.

The visual appearance of the fish fillet is a significant determinant of consumers’ purchase decisions. Depending on the rainbow trout diet, a uniform bright white or reddish/pink fillet color is desirable. Factors affecting fillet color are complex, ranging from the ability of live fish to accumulate carotenoids in the muscle to preharvest environmental conditions, early postmortem muscle metabolism, and storage conditions. Identifying genetic markers of fillet color is a desirable goal but a challenging task for the aquaculture industry. This study used weighted, single-step GWAS to explore the genetic basis of fillet color variation in rainbow trout. We identified several SNP windows explaining up to 3.5%, 2.5%, and 1.6% of the additive genetic variance for fillet redness, yellowness, and whiteness, respectively. SNPs are located within genes implicated in carotenoid metabolism (β,β-carotene 15,15′-dioxygenase, retinol dehydrogenase) and myoglobin homeostasis (ATP synthase subunit β, mitochondrial (ATP5F1B)). These genes are involved in processes that influence muscle pigmentation and postmortem flesh coloration. Other identified genes are involved in the maintenance of muscle structural integrity (kelch protein 41b (klh41b), collagen α-1(XXVIII) chain (COL28A1), and cathepsin K (CTSK)) and protection against lipid oxidation (peroxiredoxin, superoxide dismutase 2 (SOD2), sestrin-1, Ubiquitin carboxyl-terminal hydrolase-10 (USP10)). A-to-G single-nucleotide polymorphism in β,β-carotene 15,15′-dioxygenase, and USP10 result in isoleucine-to-valine and proline-to-leucine non-synonymous amino acid substitutions, respectively. Our observation confirms that fillet color is a complex trait regulated by many genes involved in carotenoid metabolism, myoglobin homeostasis, protection against lipid oxidation, and maintenance of muscle structural integrity. The significant SNPs identified in this study could be prioritized via genomic selection in breeding programs to improve fillet color in rainbow trout.

Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat.

Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1-2 nucleotides leading to non-synonymous amino acid substitutions. The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

Genetic variability in minor capsid protein (L2 gene) of human papillomavirus type 16 among Indian women.

Human papillomavirus type 16 (HPV-16) is the predominant genotype worldwide associated with invasive cervical cancer and hence remains as the focus for diagnostic development and vaccine research. L2, the minor capsid protein forms the packaging unit for the HPV genome along with the L1 protein and is primarily associated with transport of genomic DNA to the nucleus. Unlike L1, L2 is known to elicit cross-neutralizing antibodies and thus becomes a suitable candidate for pan-HPV prophylactic vaccine development. In the present study, a total of 148 cervical HPV-16 isolates from Indian women were analyzed by PCR-directed sequencing, phylogenetic analysis and in silico immunoinformatics tools to determine the L2 variations that may impact the immune response and oncogenesis. Ninety-one SNPs translating to 35 non-synonymous amino acid substitutions were observed, of these 16 substitutions are reported in the Indian isolates for the first time. T245A, L266F, S378V and S384A substitutions were significantly associated with high-grade cervical neoplastic status. Multiple substitutions were observed in samples from high-grade cervical neoplastic status as compared to those from normal cervical status (p = 0.027), specifically from the D3 sub-lineage. It was observed that substitution T85A was part of both, B and T cell epitopes recognized by MHC-I molecules; T245A was common to B and T cell epitopes recognized by MHC-II molecules and S122P/A was common to the region recognized by both MHC-I and MHC-II molecules. These findings reporting L2 protein substitutions have implications on cervical oncogenesis and design of next-generation L2-based HPV vaccines.

Impaired KIN10 function restores developmental defects in the Arabidopsis trehalose 6-phosphate synthase1 (tps1) mutant.

Summary Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6‐PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6‐phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo‐lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON‐FERMENTING1 RELATED KINASE1 (SnRK1).Here, we explored the role of SnRK1 in T6P‐mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches.We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10.Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss‐of‐function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.

Statistical modeling of SARS-CoV-2 substitution processes: predicting the next variant

We build statistical models to describe the substitution process in the SARS-CoV-2 as a function of explanatory factors describing the sequence, its function, and more. These models serve two different purposes: first, to gain knowledge about the evolutionary biology of the virus; and second, to predict future mutations in the virus, in particular, non-synonymous amino acid substitutions creating new variants. We use tens of thousands of publicly available SARS-CoV-2 sequences and consider tens of thousands of candidate models. Through a careful validation process, we confirm that our chosen models are indeed able to predict new amino acid substitutions: candidates ranked high by our model are eight times more likely to occur than random amino acid changes. We also show that named variants were highly ranked by our models before their appearance, emphasizing the value of our models for identifying likely variants and potentially utilizing this knowledge in vaccine design and other aspects of the ongoing battle against COVID-19.

Identification of Major SARS CoV-2 Variants in India using in silico PCR-RFLP analysis

PurposeAs of June 30, 2021, WHO reported 181,344,224 confirmed cases worldwide and 3,934,252 deaths due to SARS CoV-2. India accounts almost 10% of the total mortality. The mutated Variants of Concern (VoC) and Variants of Interest (VoI) has acquired non-synonymous amino acid substitutions in Spike (S), ORF1a, ORF1b, Nucleocapsid (N), Membrane (M), Envelope (E), ORF6, ORF7a, ORF3a and ORF8 regions exhibiting more virulence and higher transmission rate. The S gene displaying nucleotide polymorphisms are studied for identification of major SARS CoV-2 variants circulating in India using in silico PCR-RFLP analysis.Methods & MaterialsDNA sequences of major SARS CoV-2 variants [alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), epsilon (B.1.429+B.1.427), eta (B.1.525) and zeta (P.2)] prevailing in India retrieved from GISAID database were annotated using VIPR-VIGOR4 genome annotator tool. The S gene of variants were aligned using CLUSTALW algorithm in MEGA-X with default parameters and analyzed for sequence identity using Geneious Prime v2019.2.1. Primers were designed using Primer-BLAST tool, proof-read using FastPCR v6.7.46 and PCR-amplified using Snapgene v.3.2.1. Unique restriction sites in S amplicons of each variant were subjected to online Restriction Analyzer tool. The S amplicons were digested with restriction endonucleases and the band profile of each variant were visualized in the gel simulation tool using Snapgene v.3.2.1.ResultsThe percentage of identical sites present in S region among SARS CoV-2 variants was found to be 98.6%. The amplified products were in the length of 3,689 and 3,698 bp. Out of 400 restriction endonucleases identified, 14 buffer-compatible enzymes were selected for single-step restriction digestion to generate unique RFLP profiles for each variant. The BsaXI-XcmI-AcuI triple digest showed unique banding pattern identifiers on 2% agarose gel for individual variants including alpha, eta, delta and zeta. The ApaLI-BsaI double digest produced distinct band profiles for beta and epsilon variants.ConclusionOur study strongly suggests PCR-RFLP analysis of Spike region can differentiate major SARS CoV-2 variants that are circulating in India. Further, quadruple digestion based wet lab experiments are underway to explore the possibility of surveillance of the major variants using a single-tube reaction followed by agarose gel-based profiling.

The SARS-CoV-2 spike L452R-E484Q variant in the Indian B.1.617 strain showed significant reduction in the neutralization activity of immune sera.

To assess the impact of the key non-synonymous amino acid substitutions in the RBD of the spike protein of SARS-CoV-2 variant B.1.617.1 (dominant variant identified in the current India outbreak) on the infectivity and neutralization activities of the immune sera, L452R and E484Q (L452R-E484Q variant), pseudotyped virus was constructed (with the D614G background). The impact on binding with the neutralizing antibodies was also assessed with an ELISA assay. Pseudotyped virus carrying a L452R-E484Q variant showed a comparable infectivity compared with D614G. However, there was a significant reduction in the neutralization activity of the immune sera from non-human primates vaccinated with a recombinant receptor binding domain (RBD) protein, convalescent patients, and healthy vaccinees vaccinated with an mRNA vaccine. In addition, there was a reduction in binding of L452R-E484Q-D614G protein to the antibodies of the immune sera from vaccinated non-human primates. These results highlight the interplay between infectivity and other biologic factors involved in the natural evolution of SARS-CoV-2. Reduced neutralization activities against the L452R-E484Q variant will have an impact on health authority planning and implications for the vaccination strategy/new vaccine development.

Comparative Whole Genome Sequence Analysis and Biological Features of Clostridioides difficile Sequence Type 2‡.

Clostridioides difficile sequence type 2 (ST2) has been increasingly recognized as one of the major genotypes in China, while the genomic characteristics and biological phenotypes of Chinese ST2 strains remain to be determined. We used whole-genome sequencing and phylogenetic analysis to investigate the genomic features of 182 ST2 strains, isolated between 2011 and 2017. PCR ribotyping (RT) was performed, and antibiotic resistance, toxin concentration, and sporulation capacity were measured. The core genome Maximum-likelihood phylogenetic analysis showed that ST2 strains were distinctly segregated into two genetically diverse lineages [L1 (67.0% from Northern America) and L2], while L2 further divided into two sub-lineages, SL2a and SL2b (73.5% from China). The 36 virulence-related genes were widely distributed in ST2 genomes, but in which only 11 antibiotic resistance-associated genes were dispersedly found. Among the 25 SL2b sequenced isolates, RT014 (40.0%, n = 10) and RT020 (28.0%, n = 7) were two main genotypes with no significant difference on antibiotic resistance (χ2 = 0.024–2.667, P > 0.05). A non-synonymous amino acid substitution was found in tcdB (Y1975D) which was specific to SL2b. Although there was no significant difference in sporulation capacity between the two lineages, the average toxin B concentration (5.11 ± 3.20 ng/μL) in SL2b was significantly lower in comparison to those in L1 (10.49 ± 15.82 ng/μL) and SL2a (13.92 ± 2.39 ng/μL) (χ2 = 12.30, P < 0.05). This study described the genomic characteristics of C. difficile ST2, with many virulence loci and few antibiotic resistance elements. The Chinese ST2 strains with the mutation in codon 1975 of the tcdB gene clustering in SL2b circulating in China express low toxin B, which may be associated with mild or moderate C. difficile infection.

Emergence of transferable ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae due to a novel CMY AmpC β-lactamase in China

ObjectivesTo evaluate the molecular mechanisms of ceftazidime/avibactam (CAZ/AVI) resistance in six Klebsiella pneumoniae strains that co-produce K. pneumoniae carbapenemase (KPC)-2 and a novel variant of CMY cephalosporinase in a Chinese hospital. MethodsAntimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing (WGS) was performed to investigate potential resistance determinants. Plasmid conjugation, electroporation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) hybridization and cloning experiment were carried out to investigate the resistance plasmids and genes. ResultsA high level of CAZ/AVI resistance was observed in six KPC-Kp strains (MIC 128 mg/L). Five strains were isolated in 2015 and one in 2016, before the approval of CAZ/AVI in China. Sequence analysis indicated that all the strains belonged to sequence type (ST) 11 and uniformly carried a novel CMY AmpC β-lactamase gene, designated blaCMY-172. When compared with CMY-2, CMY-172 has a deletion of three consecutive amino acids (K290, V291 and A292) in the R2-loop region and a non-synonymous amino acid substitution at position 346 (N346I). The blaCMY-172-bearing plasmid, pKPCZA02_4, was 93.3 Kb, IncI1-I type, and conjugative; blaCMY-172 was located in an IS1294-mediated transposon. Plasmid conjugation and DNA fragment cloning proved that blaCMY-172 was responsible for CAZ/AVI resistance. ConclusionsOur study identified conjugative plasmid-mediated blaCMY-172 as a new mechanism for CAZ/AVI resistance in clinical KPC-Kp strains. Careful monitoring of CAZ/AVI susceptibility is imperative for preventing the spread of the resistance gene.

The genetic variability, phylogeny and functional significance of E6, E7 and LCR in human papillomavirus type 52 isolates in Sichuan, China

BackgroundVariations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China.MethodsLCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database.Results50 SNP sites (6 in E6, 10 in E7, 34 in LCR) were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387–7391 (TTATG) and 7698–7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287–7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions (including E3Q and K93R in the E6, and T37I, S52D, Y59D, H61Y, D64N and L99R in the E7) were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY.ConclusionThis study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.

Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

Predicted structural differences of four fertility-related Y-chromosome proteins in Macaca mulatta, M. fascicularis, and their Indochinese hybrids.

Species in the genus Macaca typically live in multimale-multifemale social groups with male macaques exhibiting some of the largest testis: body weight ratios among primates. Males are believed to experience intense levels of sperm competition. Several spermatogenesis genes are located on the Y-chromosome and, interestingly, occasional hybridization between two species has led to the introgression of the rhesus macaque (Macaca mulatta) Y-chromosome deep into the range of the long-tailed macaque (M. fascicularis). These observations have led to the prediction that the successful introgression of the rhesus Y-haplotype is due to functional differences in spermatogenesis genes compared to those of the native long-tailed Y-haplotype. We examine here four Y-chromosomal loci-RBMY, XKRY, and two nearly identical copies of CDY-and their corresponding protein sequences. The genes were surveyed in representative animals from north of, south of, and within the rhesus x long-tailed introgression zone. Our results show a series of non-synonymous amino acid substitutions present between the two Y-haplotypes. Protein structure modeling via I-TASSER revealed different folding patterns between the two species' Y-proteins, and functional predictions via TreeSAAP further reveal physicochemical differences as a result of non-synonymous substitutions. These differences inform our understanding of the evolution of primate Y-proteins involved in spermatogenesis and, in turn, have biomedical implications for human male fertility.

Deep Profiling Identifies Selection of Nonsynonymous Amino Acid Substitutions in HIV-1 Envelope During Early Infection.

Understanding the evolutionary dynamics of the viruses within an individual at or near the moment of transmission can provide critical inputs for the design of an effective vaccine for HIV infection. In this study, high-throughput sequencing technology was employed to analyze the evolutionary rate in viruses obtained at a single time point from drug-naive recently infected infants and adults in the chronic stage of disease. Gene-wise nonsynonymous (pN) and synonymous (pS) mutation rates were estimated and compared between the two groups. Significant differences were observed in the evolutionary rates between viruses in the early and late stages of infection. Higher rates of adaptive mutations in the HIV-1 envelope gene (env) were found in the chronic viruses as compared with those in the early stages of HIV infection. Conversely, percentage of nonsynonymous substitutions in env was found to be higher in recently transmitted viruses. In addition, a positive correlation was found between mutation and the evolutionary rate, and infectivity titer in recent infection. Despite the small sample size, the study identified useful information about viral evolution on transmission-associated bottlenecks. The effect of intraindividual HIV-1 evolution at the population level was highly contemporary, and the higher percentage of nonsynonymous substitutions seen in env during recent HIV-1 infection has suggested a pattern of convergent evolution leading to a positive selection for survival fitness and disease progression.