Amino Acid Variations Research Articles

Genetic diversity and antigenic variability of foot-and-mouth disease virus strains in Egypt.

Foot-and-mouth disease (FMD) continues to pose a significant threat in Egypt, necessitating thorough analyses of FMD virus (FMDV) outbreaks. This study analyzed 144 suspected FMD cases across 52 animal collections during the years 2017-2018 and 2022. Recurrent FMD outbreaks in vaccinated dairy cattle were investigated. Clinical observation, postmortem examination, and histopathological analysis were conducted. Samples were subjected to reverse transcription polymerase chain reaction (RT-PCR) using group-specific in addition to serotype-specific primers, along with direct gene sequencing of the VP1 gene from positive samples, followed by phylogenetic analysis. Signs, postmortem and histopathological lesions consistent with FMD were found. Molecular identification confirmed the presence of serotype A (G-IV topotype), O (O/EA3 topotype), and serotype SAT2 (SAT2/VII/Lib-03 topotype). All detected topotypes were distinct from currently available vaccine strains. Variations in amino acid in the epitopes of the structural protein VP1 were detected. The study highlights the genetic and antigenic variability of FMDV strains circulating in Egypt, indicating potential challenges for vaccine effectiveness.

Identification of Proteoforms Related to Nelumbo nucifera Flower Petaloid Through Proteogenomic Strategy.

Nelumbo nucifera is an aquatic plant with a high ornamental value due to its flower. Despite the release of several versions of the lotus genome, its annotation remains inefficient, which makes it difficult to obtain a more comprehensive knowledge when -omic studies are applied to understand the different biological processes. Focusing on the petaloid of the lotus flower, we conducted a comparative proteomic analysis among five major floral organs. The proteogenomic strategy was applied to analyze the mass spectrometry data in order to dig out novel proteoforms that are involved in the petaloids of the lotus flower. The results revealed that a total of 4863 proteins corresponding to novel genes were identified, with 227 containing single amino acid variants (SAAVs), and 72 originating from alternative splicing (AS) genes. In addition, a range of post-translational modifications (PTMs) events were also identified in lotus. Through functional annotation and homology analysis with 24 closely related plant species, we identified five candidate proteins associated with floral organ development, which were not identified by ordinary proteomic analysis. This study not only provides new insights into understanding the mechanism of petaloids in lotus but is also helpful in identifying new proteoforms to improve the annotation of the lotus genome.

Genomic characterization of noroviruses from an outbreak associated with oysters.

Human noroviruses are the leading cause of non-bacterial shellfish-associated gastroenteritis. In 2022, a multi-jurisdictional norovirus outbreak associated with contaminated oysters occurred that involved hundreds of illnesses. Here, we conducted genetic analysis on 30 clinical samples associated with this oyster outbreak. We first determined the capsid genotypes by Sanger sequencing and viral titers by droplet-digital reverse transcription PCR. Multiple genotypes were identified in this outbreak, which could indicate contamination with wastewaters. The majority of samples belonged to GII.3[P12], followed by GII.2[P16], GII.17[P17], and GII.4 Sydney[P16]. We next proceeded with whole-genome sequencing and obtained full genomes for 19 samples. Phylogenetic analysis revealed that some of the isolates showed high similarity with the sequences isolated from the United States related to the same outbreak. We also analyzed amino acid variations in the sequenced genomes and found that overall the GII.3[P12] isolates have lower variations compared to other genotypes.IMPORTANCENorovirus outbreaks associated with contaminated shellfish occur frequently. Whole-genome sequencing (WGS) could play a critical role in understanding and controlling norovirus outbreaks as it allows for source attribution, tracking transmission pathways, and detecting recurrent or linked outbreaks. Here, we described how the data obtained by WGS were employed for understanding transmission patterns and norovirus epidemiology.

The Functional Identification of the CYP2E1 Gene in the Kidney of Lepus yarkandensis.

This study aims to identify the function of the cytochrome P450 2E1 (CYP2E1) gene in the kidneys of Lepus yarkandensis. CYP2E1 is a significant metabolic enzyme involved in the metabolism of various endogenous and exogenous compounds and is associated with the occurrence and progression of multiple diseases. Given L. yarkandensis's ability to survive in the extremely arid L. yarkandensis, we hypothesize that CYP2E1 in its kidneys plays a crucial role in adaptability. Through molecular cloning and sequence analysis, we discovered that the CYP2E1 gene of Lepus yarkandensis encodes a protein of 493 amino acids. The 493-amino acid protein encoded by the Lepus yarkandensis CYP2E1 gene shows 13 amino acid variation sites compared to the homologous protein in Oryctolagus cuniculus. The protein is primarily localized to the endoplasmic reticulum membrane and lacks transmembrane structures. In the yeast expression system, the heterologous expression of the CYP2E1 gene enhanced the yeast's tolerance to drought, salinity, and high temperatures, achieved by increasing antioxidant enzyme activity and reducing levels of oxidative stress markers. Additionally, this study identified a "Yeast Oxidative Stress Lethal Threshold (Yeast OSLT)" under specific stress conditions. Once this threshold is exceeded, the cell's antioxidant defense system can no longer maintain cellular homeostasis, leading to massive cell death. Although CYP2E1 did not change this threshold, it contributed to cell survival to some extent. These findings not only reveal the function of L. yarkandensis CYP2E1 in stress adaptation but also provide valuable molecular insights into its survival strategy in extreme environments.

Amino Acids Frequency and Interaction Trends: Comprehensive Analysis of Experimentally Validated Viral Antigen-Antibody Complexes.

Antibodies have specific binding capabilities and therapeutic potential for treating various diseases, including viral infections. The amino acid composition of the hypervariable complementarity determining regions (CDR) loops and the framework regions (FR) are the determining factors for the affinity and therapeutic efficacy of the antibodies. In this study selected and curated, 77 viral antigen-human antibody complexes from Protein data bank from the Thera-SAbdab database were analyzed. The results revealed diversity indices within specific CDR regions, amino acid frequencies, paratope-epitope interactions, bond formations, and bond types among the analyzed viral Ag-Ab complexes. The finding revealed that Ser, Gly, Tyr, Thr, and Phe are prominently present in the antibody CDRs. Analysis of CDR profiles indicated an average amino acid diversity of 60-80% in heavy chain CDRs and 45-60% in light chain CDRs. Aromatic residues, particularly Tyr, Phe, and Trp showed significant involvement in the paratope-epitope interactions in the heavy chain, while Tyr, Ser, and Thr were key contributors in the light chain. Furthermore, the study examined the occurrence of amino acids in both light and heavy chains of viral Ag- human Ab complexes, analyzing the presence of amino acids as single residues, dipeptides and tripeptides. The analysis is crucial for enhancing the antibody engineering processes including, design, optimization, affinity enhancement, and overall antibody development.

First detection of the S989P+V1016G+D1763Y haplotype and expansion of voltage-gated sodium channel mutations in Aedes aegypti in Taiwan in 2016-2023.

Aedes aegypti transmits various arthropod-borne diseases such as dengue, posing a significant burden to public health in tropical and subtropical regions. Pyrethroid-based control strategies are effective in managing this vector; however, the development of insecticide resistance has hindered these efforts. Hence, long-term monitoring of insecticide resistance in mosquito populations is crucial for effective vector and disease control. In this study, we identified insecticide resistance due to a voltage-gated sodium channel (vgsc) mutation in Ae. aegypti in Taiwan between 2016 and 2023. In total, 1,761 field-caught Ae. aegypti samples from Tainan, Kaohsiung, and Pingtung were genotyped. The frequencies of S989P, V1016G, T1520I, F1534C, and D1763Y amino acid variants increased over the surveillance period. A T1520I mutation was detected for the first time and has since rapidly spread throughout Taiwan. The triple-mutant haplotype PGTFY was first documented in Ae. aegypti. Moreover, the unmutated haplotype vanished in Taiwan, suggesting that the vgsc mutations were fixed in local populations of Ae. aegypti. Five resistance-associated genotypes, SVTCD/SVTCD, SGTFY/PGTFD, SVTCD/SGTFY, PGTFD/PGTFD, and SVTCD/PGTFD, exhibited an increased frequency and accounted for 76% of the total field population. We also detected the resistant genotype SVICD/PGTFD, and its frequency increased 13-fold in the field between 2016 and 2023. Moreover, we also observed that mutations differed geographically, with S989P mainly found in Kaohsiung and V1016G in Kaohsiung and Pingtung. The frequency of T1520I was noticeably higher in Kaohsiung, and D1763Y occurred mainly in Tainan. The emergence and expansion of mutations along with the disappearance of wild-type mosquitoes in Taiwan underscores the threat of resistance and difficulty of mosquito control in Taiwan as well as globally. This study determined the insecticide resistance status of Ae. aegypti in Taiwan, and the findings will be helpful for resistance monitoring in areas where pyrethroids are used to control Ae. aegypti.

Hepatitis B virus genotypes A1 and A2 have distinct replication phenotypes due to polymorphisms in the HBx gene.

HBV genotype A has two major subtypes, A1 (commonly in Africa) and A2 (commonly in Europe) with only 4% nucleotide differences. Individuals infected with these two subtypes appear to have different clinical manifestations and virologic features. Whether such a difference results from the virus or host has not been established. Using HBV generated from molecule clones of subtypes A1 and A2 in cell culture (HBVcc), we demonstrate that HBVcc of subtypes A1 and A2 can be passaged in vitro and in vivo and respond equally well to human IFN-α treatment. HBVcc passaged in human liver chimeric mice (HBVmp) infected human hepatocytes more efficiently than that of the original HBVcc. Subtype A2 showed a much higher viral replication level than that of subtype A1. Mechanistic investigations using constructs with chimeric A1/A2 sequences and specific mutations indicated that subtype A2 has an inherently higher replication phenotype due to specific polymorphisms in the HBx gene resulting in amino acid variations. Studies of HBx expression demonstrated that A1 HBx is expressed at a much lower level than that of A2 HBx. Mutagenesis studies identified two HBx amino acid variations responsible for the observed phenotypic difference. Using AlphaFold2, we generated structural models of HBx proteins of A1 and A2. Superposition of the two models reveal that the overall structural motifs are similarly aligned, except for the C-terminal peptides diverging between the A1 and A2 models, possibly explaining their functional difference. In conclusion, using various in vitro and in vivo models, here we show that subtype A2 has an inherently higher replication phenotype due to polymorphisms in HBx that result in possible differences in structure and expression level of the two subtype HBx proteins. This genotypic difference potentially explains the reported clinical differences between the two subtypes as well as providing a previously unrecognized association between viral sequence variations and clinical manifestations of HBV infection in humans.

Rapid Synthesis of Novel Pyrazino-Pyrido-Pyrimidinones Derived from Fumiquinazoline Alkaloids

2,5-Diketopiperazines (2,5-DKPs) are valued in Medicinal Chemistry for their diverse properties and are considered privileged structures. The natural alkaloids class of fumiquinazolines, combining 2,5-DKP and quinazolinone cores within a pyrazino[1,2-b]quinazoline-3,6-dione framework, is known for its broad biological activity. Although few synthetic approaches to such structures have been explored, this study focuses on synthesizing novel fumiquinazolines analogs featuring a pyrazinopyrido[2,3-d]pyrimidine-5,7-dione core bearing various substituents on the pyrazine moiety (C-6 and C-9), classified into three series (A, B, and C). To investigate an efficient sequential one-pot method, we used a pyridine precursor and L-amino acids in two condensation and cyclization reactions, under controlled microwave irradiations at lower temperatures than previously reported. This approach generated protected bicyclic intermediates, particularly pyrido[2,3-d]pyrimidin-4(3H)-one derivatives, which were smoothly deprotected, allowing for final intramolecular cyclization to yield disubstituted fumiquinazoline analogs (series A) without observed epimerization. The optimized protocol revealed greater efficiency for monosubstituted analogs (series B and C), speeding up the tricyclic products synthesis without the need for intermediates isolation and deprotection. Overall, this study demonstrates the incorporation of diverse protected amino acids, featuring different electronic and structural side chains, via a rapid and effective sequential one-pot strategy, resulting in the synthesis of 19 novel fumiquinazoline analogs.

Decoding the blueprint of receptor binding by filoviruses through large-scale binding assays and machine learning.

Evidence suggests that bats are important hosts of filoviruses, yet the specific species involved remain largely unidentified. Niemann-Pick C1 (NPC1) is an essential entry receptor, with amino acid variations influencing viral susceptibility and species-specific tropism. Herein, we conducted combinatorial binding studies with seven filovirus glycoproteins (GPs) and NPC1 orthologs from 81 bat species. We found that GP-NPC1 binding correlated poorly with phylogeny. By integrating binding assays with machine learning, we identified genetic factors influencing virus-receptor-binding and predicted GP-NPC1-binding avidity for additional filoviruses and bats. Moreover, combining receptor-binding avidities with bat geographic distribution and the locations of previous Ebola outbreaks allowed us to rank bats by their potential as Ebola virus hosts. This study represents a comprehensive investigation of filovirus-receptor binding in bats (1,484 GP-NPC1 pairs, 11 filoviruses, and 135 bats) and describes a multidisciplinary approach to predict susceptible species and guide filovirus host surveillance.

Variability in HIV-1 transmitted/founder virus susceptibility to combined APOBEC3F and APOBEC3G host restriction.

Several APOBEC3 enzymes restrict HIV-1 by deaminating cytosine to form uracil in single-stranded proviral (-)DNA. However, HIV-1 Vif counteracts their activity by inducing their proteasomal degradation. This counteraction by Vif is incomplete, as evidenced by footprints of APOBEC3-mediated mutations within integrated proviral genomes of people living with HIV-1. The relative contributions of multiple APOBEC3s in HIV-1 restriction are not fully understood. Here, we investigated the activity of co-expressed APOBEC3F and APOBEC3G against HIV-1 Subtype B and Subtype C transmitted/founder viruses. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain. We provide evidence that this results in protection of APOBEC3F from Vif-mediated degradation because the APOBEC3F N-terminal domain contains residues required for recognition by Vif. We also found that HIV-1 Subtype C Vifs, but not Subtype B Vifs, were less active against APOBEC3G when APOBEC3F and APOBEC3G were co-expressed. Consequently, when APOBEC3F and APOBEC3G were expressed together in a single cycle of HIV-1 replication, only HIV-1 Subtype C viruses showed a decrease in relative infectivity compared to when APOBEC3G was expressed alone. Inspection of Vif amino acid sequences revealed that differences in amino acids adjacent to conserved sequences influenced the Vif-mediated APOBEC3 degradation ability. Altogether, the data provide a possible mechanism for how combined expression of APOBEC3F and APOBEC3G could contribute to mutagenesis of HIV-1 proviral genomes despite the presence of Vif and provide evidence for variability in the Vif-mediated APOBEC3 degradation ability of transmitted/founder viruses.IMPORTANCEAPOBEC3 enzymes suppress HIV-1 infection by inducing cytosine deamination in proviral DNA but are hindered by HIV-1 Vif, which leads to APOBEC3 proteasomal degradation. Moving away from traditional studies that used lab-adapted HIV-1 Subtype B viruses and singular APOBEC3 enzymes, we examined how transmitted/founder isolates of HIV-1 replicated in the presence of APOBEC3F and APOBEC3G. We determined that APOBEC3F interacts with APOBEC3G through its N-terminal domain and that APOBEC3F, like APOBEC3G, has Vif-mediated degradation determinants in the N-terminal domain. This enabled APOBEC3F to be partially resistant to Vif-mediated degradation. We also demonstrated that Subtype C is more susceptible than Subtype B HIV-1 to combined APOBEC3F/APOBEC3G restriction and identified Vif variations influencing APOBEC3 degradation ability. Importantly, Vif amino acid variation outside of previously identified conserved regions mediated APOBEC3 degradation and HIV-1 Vif subtype-specific differences. Altogether, we identified factors that affect susceptibility to APOBEC3F/APOBEC3G restriction.

Epidemiological Investigation and Genetic Analysis of Duck Circovirus in Korea from 2013 to 2022.

Duck circovirus (DuCV) infections cause immunosuppression in ducks, potentially leading to significant economic losses for the duck farming industry. This study investigates the prevalence, genetic characteristics, and evolutionary trends of DuCV in Korea between 2013 and 2022. Samples from 184 farms across seven provinces were analyzed using polymerase chain reaction (PCR). The overall DuCV infection rate was 29.4% (54/184), with Jeollanam-do showing the highest prevalence (37.5%, 15/40). Ducks aged 3-6 weeks were most susceptible to infection, while ducklings younger than one week were rarely infected. Whole-genome sequencing was performed on 24 positive samples with phylogenetic analysis revealing that DuCV-1b is the predominant subtype in Korea (23/24 strains). Notably, a Korean DuCV-1a subtype strain was identified for the first time, showing close genetic relatedness to Chinese DuCV 1a strains. Novel subtype-specific amino acid variations in ORF1 and ORF2 were statistically analyzed and classified. Recombination analysis suggested some Korean DuCV-1b strains may have resulted from recombination events involving strains from different countries. This comprehensive study provides crucial insights into the current prevalence, genetic diversity, and evolutionary dynamics of DuCV in Korea, offering valuable data for developing effective control strategies and understanding the global epidemiology of this economically important avian pathogen.

Lipid Trafficking in Diverse Bacteria.

ConspectusLipids are essential for life and serve as cell envelope components, signaling molecules, and nutrients. For lipids to achieve their required functions, they need to be correctly localized. This requires the action of transporter proteins and an energy source. The current understanding of bacterial lipid transporters is limited to a few classes. Given the diversity of lipid species and the predicted existence of specific lipid transporters, many more transporters await discovery and characterization. These proteins could be prime targets for modulators that control bacterial cell proliferation and pathogenesis.One overarching goal of our research is to understand the molecular mechanisms of bacterial metabolite trafficking, including lipids, and to leverage that understanding to identify or engineer inhibitory ligands. In recent years, our work has revealed two novel lipid transport systems in bacteria: bacterial sterol transporters (Bst) A, B, and C in Methylococcus capsulatus and the TatT proteins in Enhygromyxa salina and Treponema pallidum. Both systems are composed of transporters bioinformatically identified as being involved in the transport of other metabolites, but substrates were never revealed. However, the genetic colocalization of the genes encoding BstABC with sterol biosynthetic enzymes in M. capsulatus suggested that they might recognize sterols as substrates. Also, homologues of TatTs are present in diverse bacteria but are overrepresented in bacteria deficient in de novo lipid synthesis or residing in nutrient-poor environments; we reasoned that these proteins might facilitate the transport of lipids. Our efforts to reveal the substrate scope of two TatT proteins revealed their engagement with long-chain fatty acids.Enabling the discovery of the BstABC system and the TatT proteins were bioinformatic analyses, quantitative measurements of protein-ligand equilibrium affinities, and high-resolution structural studies that provided remarkable insights into ligand binding cavities and the structural basis for ligand interaction. These approaches, in particular our bioinformatics and structural work, highlighted the diversity of protein sequence and structures amenable to lipid engagement. These observations allowed the hypothesis that lipid handling proteins, in general and especially so in the bacterial domain, can have diverse amino acid compositions and three-dimensional structures. As such, bioinformatics geared at identifying them in poorly characterized genomes is likely to miss many candidates that diverge from well-characterized family members.This realization spurred efforts to understand the unifying features in all of the lipid handling proteins we have characterized to date. To do this, we inspected the ligand binding sites of the proteins: they were remarkably hydrophobic and sometimes displayed a dichotomy of hydrophobic and hydrophilic amino acids, akin to the ligands that they accommodate in those cavities. Because of this, we reasoned that the physicochemical features of ligand binding cavities could be accurate predictors of a protein's propensity to bind lipids. This finding was leveraged to create structure-based lipid-interacting pocket predictor (SLiPP), a machine-learning algorithm capable of identifying ligand cavities with physico-chemical features consistent with those of known lipid binding sites. SLiPP is especially useful in poorly annotated genomes (such as with bacterial pathogens), where it could reveal candidate proteins to be targeted for the development of antimicrobials.

Exploring the impact of variations in the mucolipin1 protein that result in mucolipidosis type 4 using the technique of molecular docking and dynamics simulation

Mucolipidosis type IV (MLIV) is classified as an exceptionally autosomal recessive condition due to a change in MCOLN1 that encodes the mucolipin-1 protein. ML-1 is a membrane protein comprising 6 Trans regions, which are situated at the LELs, a serine lipase area, and a nuclear localization sign. The characteristic features of the ML4 patients are mental retardation and skeletal deformities due to an increase in lipid molecules in the brain, other tissues, and organs. The fundamental goal of the work is to identify the most significant amino acid variants via a computational pipeline. The twenty-three amino acid variants that are responsible for the condition were retrieved from the public domain: L106P and L447P amino acid variants, with the following categories: extremely conserved, highly pathogenic, most interfering with protein function, more structurally unstable, and having promising Phenotyping characteristics was scrutinized from the series of bioinformatics tools that denote its significant nature. A docking and dynamics study was initiated to identify the interaction profiling and interatomic simulation between the Native, L106P, and L447P and the ligand ML-SA1 (it was known to ease the fatty acid buildup in lysosomes of cellular models of Mucolipidosis type IV) and had a score of −6.19, −5.12, and −5.21 kcal/mol, followed by a duplicate 100-ns run trajectory results, which assisted in detecting the stable nature of all the complex structures. Hence, this work helps to recognize the significant role of the scrutinized amino acid variants and, on the other hand, the stable nature of the ligand using standard computational tools.

Proline-Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β-Casein Containing Milks.

The proline amino acid and prolyl residues of peptides/proteins confer unique biological and biochemical properties that motivates the development of proline-selective analysis. The study focuses on one specific class of problem, the detection of single amino acid variants involving proline, and reports a Pro-selective electrochemiluminescence (ECL) method. To develop this method, the A1-/A2- variants of milk's β-casein protein are investigated because it is a well-established example and abundant samples are readily available. Specifically, β-casein has 209 amino acids with 34 (or 35) proline residues: the A1-variant has a Pro-to-His substitution at position 67 (relative to the A2 variant). The study shows that proline's strong luminescence allows the generic discrimination of: Pro from other amino acids; an A2-oligopeptide from an A1-oligopeptide; the A2-β-casein variant from the A1-variant; and commercially-available A2 milks from A1-containing regular milks. The evidence indicates that luminescence depends on proline content and accessibility, as well as signal quenching. Compared to conventional immunoassays, the ECL method is simple, rapid, and inexpensive. Further, the ECL-method is Pro-selective (vs molecularly-selective like typical immunoassays) which should make it broadly useful for studying the role of proline in biology and especially useful for tracking the digestion of proline-rich proteins in the diet.

Distinct amino acid substitutions in the EEV glycoprotein and DNA-dependent RNA polymerase of lumpy skin disease virus identified in wetland areas of Bangladesh.

The recent outbreak of lumpy skin disease virus (LSDV) in the wetland areas of Bangladesh presents a significant concern for both animal health and regional biosecurity. Epidemiological investigations into nine major outbreaks in the wetland areas revealed distinctive clinical symptoms in affected cattle, including elevated body temperature, excessive salivation, and the presence of skin nodules. Histopathological examination unveiled larger nodules compared to previous outbreaks, along with signs of secondary infection. Molecular analysis confirmed the presence of LSDV in all samples, with subsequent sequencing revealing genetic similarities with virus isolates of Bangladesh, India, China, Russia, Serbia and Greece. Most importantly amino acid variations in the viral EEV glycoprotein and DNA-dependent RNA polymerase were revealed that also altered the structures of the respective proteins significantly suggesting potential implications for viral pathogenesis. Additionally, successful isolation of LSDV in Vero cells demonstrated cytopathic effects, supporting the potential for vaccine development. In conclusion, this study provides comprehensive insights into the epidemiology, genomic characters with altered predicted structures of two major viral proteins and pathogenesis of LSDV outbreaks in Bangladesh. These findings emphasize the critical need for ongoing monitoring and adaptive control strategies, including the development of effective vaccines, to mitigate the impact of LSDV in affected regions and safeguard regional livestock health.

NS2B-D55E and NS2B-E65D Variations Are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model.

Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at the internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging the NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in the regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residues 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses.

Chemical quantification of N-acyl alanine methyl ester (NAME) production and impact on temporal gene expression patterns in Roseovarius tolerans EL-164

BackgroundPrevious studies have identified structurally diverse N-acyl amino acid methyl esters (NAMEs) in culture extracts of Roseovarius tolerans EL-164 (Roseobacteraceae). NAMEs are structural analogues of the common signaling compounds N-acyl homoserine lactones (AHLs), but do not participate in AHL-mediated signaling. NAMEs show minor antialgal and antimicrobial activity, but whether this activity serves as the primary ecological role remains unclear.ResultsTo enable dose-dependent bioactivity-testing, we have established a chromatographic method for quantification of NAMEs in bacterial culture extracts. The concentrations determined for the two major NAMEs produced by EL-164, C16:1-NAME and C17:1-NAME, ranged between 0.685 and 5.731 mg L− 1 (2.0-16.9 µM) and 5.3–86.4 µg L− 1 (15.0-244.3 nM), respectively. Co-quantification of the C14:1-AHL showed concentrations ranging between 17.5 and 58.7 mg L− 1 (56.6-189.7 µM). We observed distinct production patterns for NAMEs and AHLs, with a continuous NAME production during the entire incubation period. We conducted a spike-in experiment, using the determined metabolite concentrations. By comparing the transcriptomes of pre- and post-metabolite-spikes, we identified three clusters of differentially expressed genes with distinct temporal expression patterns. Expression levels of stress response genes differed between NAME- and AHL-spiked EL-164 cultures in the stationary phase.ConclusionsOur findings support previous studies suggesting an ecological role for C16:1-NAME as antibiotic, by proving that NAME concentrations in batch cultures were higher than the minimal inhibitory concentrations against Maribacter sp. 62 − 1 (Flavobacteriia) and Skeletonema costatum CCMP 1332 (Coscinodiscophyceae) reported in the literature. Our study further exemplified the broad application range of dose-dependent testing and highlighted the different biological activities of NAMEs and AHLs.

Unnatural Cyclopeptide Synthesis via Cu-Catalyzed 1,3-Dipolar Cycloaddition of Azomethine Ylides.

Cyclic peptides are valued synthetic targets in organic and medicinal chemistry. Herein, we report an efficient strategy for the synthesis of unnatural cyclic peptides via the Cu-catalyzed 1,3-dipolar cycloaddition of azomethylene ylides. Linear precursors of different lengths and bearing diverse amino acids (26 examples) are shown to be compatible with this method, affording good yields and complete endo-diastereoselectivities. Density functional theory (DFT) calculations support a stepwise mechanism in which Cu plays a key role in the preorganization of the reactants.

Effect of Incorporating Fish Protein Hydrolysate into Pasta: Impact on Protein Digestibility, Starch Digestibility, Amino Acid, and Fatty Acid Profiles

The incorporation of fish protein hydrolysate (FPH) with the concentration of 5, 10, 15 and 20% prepared from fish waste using 1% pepsin enzyme was found beneficial in semolina-based pasta. The In-vitro protein and starch digestibility of the FPH incorporated pasta were studied. Besides, amino acid and fatty acid composition of the pasta were also evaluated. The protein digestibility of the pasta was in the range of 80.32% to 83.76% and digestibility curve was generated by plotting the pH and time. The value of sugar released at 20 min, 60 min and 120 min was recorded and a graph was developed showing readily digestible starch (RDS) and slowly digestible starch (SDS). The pasta's diverse amino acid profile, including 8 essentials and 9 non-essentials, alongside saturated fats at 39.93%-44.65% and PUFA at 12.49%-16.27%, collectively define its nutritional quality.

Amino acid variability at W194 of Staphylococcus aureus sortase A alters nucleophile specificity.

Bacterial sortases are a family of cysteine transpeptidases in Gram-positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase-mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β-carbon by W194, a tryptophan located in the β7-β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET-based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D-Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D-Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase-mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies.