Amino Acid Sequence Alignment Research Articles

Analysis of Cotton Leafroll Dwarf Virus P0 Gene Sequences from South Carolina Reveals Low Variability Among Isolates.

Cotton leafroll dwarf virus (CLRDV) is emerging across the major cotton-producing states of the southern United States. Because it was detected in nearly all cotton-producing states within a few years of its initial detection in the United States, the spread of the virus has apparently occurred rapidly. In this study spanning three growing seasons in South Carolina, we collected CLRDV isolates from symptomatic and asymptomatic cotton plants in 10 counties. The genomic region encoding P0, the viral suppressor of RNA silencing, was sequenced and compared among CLRDV isolates. Low variability among CLRDV P0 sequences from South Carolina isolates with similarities to other United States isolates was revealed by amino acid sequence alignment and phylogenetic analysis. Low variability among South Carolina isolates was also confirmed by sequencing a subset of eight near-complete genomes of CLRDV isolates. Although sequence variability was low among South Carolina isolates, this data should be taken in the context of all United States isolates, for which diversity may be higher than initially expected. Sequences gathered in this study add to the body of knowledge on CLRDV diversity in the United States.

The isolation and functional identification of a phosphoenolpyruvate carboxykinase gene from Saccharina japonica

Similar to other macroalgae, Saccharina japonica has CO2-concentrating mechanism (CCM) that allows high photosynthesis efficiency and elevates biomass. Phosphoenolpyruvate carboxykinase (PEPCK) in cytoplasm is an essential component of CCM. However, no reports on cytosolic PEPCK of S. japonica are available. In this study, the full-length cDNA sequence of a PEPCK gene (SjPCK1) predicted to be localized in cytoplasm, was screened from the full-length transcriptome of S. japonica gametophytes and identified by RT-PCR. The complete cDNA sequence of SjPCK1 was 2174 bp in length, which encompassed an open reading frame (ORF) of 1734 bp, a 5′- untranslated region (UTR) of 216 bp and a 3′-UTR of 224 bp. In parallel, the genomic DNA of SjPCK1 was 21 294 bp in length, characterized by the presence of 11 introns and 12 exons. It encoded a protein of 577 amino acids with a molecular weight of 63 kD and an isoelectric point of 8.47. Amino acid sequence alignment showed that the functional sites of PEPCK were highly conserved in the selected species. Phylogenetic analysis and sequence characterization showed that SjPCK1 was an ATP-dependent PEPCK. SjPCK1 gene was expressed by using Escherichia coli prokaryotic expression system, and the SjPCK1 protein with His 6 tag (rSjPCK1) was 81.93 kD in molecular weight. Enzyme activity assay results showed that the specific activity of carboxylase and decarboxylase of rSjPCK1 was 1.91 ± 0.29 U/mg prog and 11.3 ± 1.97 U/mg prog, respectively. These findings provide valuable molecular and biochemical insights for a further analysis of the role of cytosolic PEPCK in the storage of inorganic carbon in S. japonica.

Molecular Epidemiology and Genetic Diversity of Human Respiratory Syncytial Virus in Sicily during Pre- and Post-COVID-19 Surveillance Seasons.

Human respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection of global significance. In this study, we investigated the molecular epidemiology and the genetic variability of hRSV over seven surveillance seasons between 2015 and 2023 in Sicily, Italy. hRSV subgroups co-circulated through every season, although hRSV-B mostly prevailed. After the considerable reduction in the circulation of hRSV due to the widespread implementation of non-pharmaceutical preventive measures during the COVID-19 pandemic, hRSV rapidly re-emerged at a high intensity in 2022-2023. The G gene was sequenced for genotyping and analysis of deduced amino acids. A total of 128 hRSV-A and 179 hRSV-B G gene sequences were obtained. The phylogenetic analysis revealed that the GA2.3.5a (ON1) and GB5.0.5a (BA9) genotypes were responsible for the hRSV epidemics in Sicily.; only one strain belonged to the genotype GB5.0.4a. No differences were observed in the circulating genotypes during pre- and post-pandemic years. Amino acid sequence alignment revealed the continuous evolution of the G gene, with a combination of amino acid changes specifically appearing in 2022-2023. The predicted N-glycosylation sites were relatively conserved in ON1 and BA9 genotype strains. Our findings augment the understanding and prediction of the seasonal evolution of hRSV at the local level and its implication in the monitoring of novel variants worth considering in better design of candidate vaccines.

The Radical SAM Heme Synthase AhbD from Methanosarcina barkeri Contains Two Auxiliary [4Fe-4S] Clusters.

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron-sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron-sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer.

Heterozygous RELN missense variants associated with genetic generalized epilepsy

PurposeThe RELN gene encodes the secreted glycoprotein Reelin and has important functions in both developing and adult brains. In this study, we aimed to explore the association between the RELN and genetic generalized epilepsy (GGE). MethodsWe performed whole-exome sequencing on a cohort of 92 patients with GGE. Based on amino acid sequence alignments, allele frequency, pedigree validation and computational modeling, the RELN variants were identified and clinical features of cases were summarized. Cell-based Reelin secretion assays were examined by Western blotting. Alterations of mutant Reelin transport through the secretion pathway were detected by immunofluorescence staining. ResultsThree novel pathogenic RELN variants (3.26%; c.2260C>T/p.R754W, c.2914C>G/p.P972A and c.3029G>A/p.R1010H) were identified. All probands showed adolescence-onset generalized seizures characterized by generalized epileptiform discharges with normal EEG backgrounds, no or mild cognitive impairment, and responded well to anti-seizure medications. All these variants were located in the central regions from 1B to 2A consecutive repeats, and protein modeling demonstrated structural alterations in Reelin. Moreover, we found that these heterozygous missense variants significantly decreased the secretion of mutant proteins in HEK-293T cells, and this impairment was due to the altered transport of mutant Reelin in the secretion pathway. ConclusionThese results suggest that RELN is potentially associated with GGE. The phenotype of GGE caused by RELN variants is relatively mild, and the pathogenic mechanism may involve a loss-of-function.

Functional expression of an antimicrobial peptide, belonging to halocin C8 family, from Natrinema sp. RNS21 in Escherichia coli.

Halocins, the proteinaceous antimicrobial agents produced by haloarchaea, may be used for the preservation of salted foods and the treatment of diseases. For their application and function explanation, it is necessary to produce the active recombinants. In this work, a haloarchaeal strain producing halocin was isolated from the salt-fermented shrimp and identified as Natrinema sp. RNS21 by 16S rRNA gene sequence analysis. From 1 L of RNS21 culture, about 0.32mg of halocin with 96% purity was obtained. Based on the molecular weight, stability and amino acid sequence alignment, the antimicrobial peptide belonged to the halocin C8 (HalC8) family. HalC8 was expressed by fusion with glutathione-S-transferase (GST) in E. coli, followed by affinity purification and enterokinase (EK) cleavage. About 6.2mg of recombinant HalC8 with 95% purity was obtained from 1 L of E. coli culture. MALDI-TOF-MS and RP-HPLC analysis indicated that the molecular weight and folding pattern of purified recombinant HalC8 were the same as those of native HalC8. Recombinant HalC8 showed obvious inhibitory activity against Haloferax volcanii. Contrast to native HalC8, the active recombinant HalC8 could be easily produced in a short time with a high yield.

Stimulator of interferon genes from Asian swamp eel (MaSTING) is involved in host defense against bacterial infection.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated protein that plays critical roles in innate immunity and pathogenesis of various diseases. To date, teleost STING against viral stimulation has been identified, whereas STING signaling events in fish against bacteria are not well understood. In the present study, the open reading frame (ORF) of STING from Asian swamp eel (Monopterus albus) was cloned (named MaSTING) and its roles in bacterial infection were investigated. Amino acid sequence alignment and phylogenetic analysis revealed that MaSTING had conserved structures with mammalian STING and shared the closest relationship with mandarin fish STING. Subcellular localization analysis showed that MaSTING distributed in the whole cytoplasm and mainly co-localized with ER. Expression pattern analysis found that MaSTING was constitutively expressed in all the examined tissues with the highest expression in the liver and spleen. Post stimulation with bacteria and various PAMPs, the expression of MaSTING was induced at indicated time points in the immune-related organs and isolated peripheral blood leucocytes. Furthermore, the mechanism underlying MaSTING against bacterial infection was further studied. The qPCR analysis showed that MaSTING overexpression promoted 2'3'-cGAMP induced the expression of IFN-1, ISG15, Viperin, Mx, IL-1β and TNF-α. Western blotting assay suggested that MaSTING significantly enhanced the phosphorylation of TANK-binding kinase 1 (TBK1) and p65. MaSTING also significantly increased the luciferase activity of IFN-1 and NF-κB promoters. Taken together, MaSTING is involved in host defense against bacterial infection by inducing the inflammatory response.

Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

An amino acid substitution in HCV core antigen limits its use as a reliable measure of HCV infection compared with HCV RNA.

Hepatitis C virus (HCV) is a viral pathogen that causes chronic hepatitis, which can lead to cirrhosis and hepatocellular carcinoma. Detection of HCV RNA is the standard method used to diagnose the disease and monitor antiviral treatment. A quantification assay for the HCV core antigen (HCVcAg) has been proposed as a simplified alternative to the HCV RNA test for predicting active HCV infection, with the aim of achieving the global goal of eliminating hepatitis. The objective of this study was to determine the correlation between HCV RNA and HCVcAg, as well as the impact of amino acid sequence heterogeneity on HCVcAg quantification. Our findings demonstrated a strong positive correlation between HCV RNA and HCVcAg across all HCV genotypes (1a, 1b, 3a, and 6), with correlation coefficients ranging from 0.88 to 0.96 (p < 0.001). However, in some cases, samples with genotypes 3a and 6 exhibited lower HCVcAg levels than expected based on the corresponding HCV RNA values. Upon the core amino acid sequence alignment, it was observed that samples exhibiting low core antigen levels had an amino acid substitution at position 49, where threonine was replaced by either alanine or valine. Core mutation at this position may correlate with one of the epitope regions recognized by anti-HCV monoclonal antibodies. The present findings suggest that the utilization of HCVcAg as a standalone marker for HCV RNA might not provide adequate sensitivity for the detection of HCV infection, especially in cases where there are variations in the amino acid sequence of the core region and a low viral load of HCV RNA.

Evaluation of transmembrane domain deletions on hyaluronic acid polymerization of hyaluronan synthase isolated from Streptococcus equisimilis group G.

The membrane enzyme of hyaluronan synthase (HAS) is the key enzyme in hyaluronic acid (HA) biosynthesis by coupling UDP-sugars. Prior studies proposed the C-terminus region of HAS enzyme mediates the production rate and molecular weight of HA. The current study describes the isolation and characterizations of a transmembrane HAS enzyme isolated from Streptococcus equisimilis Group G (GGS-HAS) in vitro. The effect of transmembrane domains (TMDs) on HA productivity was determined and the shortest active variant was also identified by recombinant expression of full-length and five truncated forms of GGS-HAS in Escherichia coli. We found that the GGS-HAS enzyme is longer than that of S. equisimilis group C (GCS-HAS) which includes three more residues (LER) at the C-terminus region (positions 418-420) and also one-point mutation at position 120 (E120D). Amino acid sequence alignment demonstrated 98% and 71% identity of GGS-HAS with that of S. equisimilis Group C and S. pyogenes Group A, respectively. The in vitro productivity of the full-length enzyme was 35.57µg/nmol, however, extended TMD deletions led to a reduction in the HA productivity. The HAS-123 variant showed the highest activity among the truncated forms, indicating the essential role of first, second, and third TMDs for the full activity. Despite a decline in activity, the intracellular variant can still mediate the binding and polymerization of HA without any need for TMDs. This significant finding suggests that the intracellular domain is the core for HA biosynthesis in the enzyme and other domains are probably involved in other attributes including the enzyme kinetics that affect the size distribution of the polymer. However, more investigations on the recombinant forms are still needed to confirm clearly the role of each transmembrane domain on these properties.

In Silico Identification of Potential Inhibitors of the SARS-CoV-2 Main Protease among a PubChem Database of Avian Infectious Bronchitis Virus 3CLPro Inhibitors.

Remarkable structural homologies between the main proteases of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the avian infectious bronchitis virus (IBV) were revealed by comparative amino-acid sequence and 3D structural alignment. Assessing whether reported IBV 3CLPro inhibitors could also interact with SARS-CoV-2 has been undertaken in silico using a PubChem BioAssay database of 388 compounds active on the avian infectious bronchitis virus 3C-like protease. Docking studies of this database on the SARS-CoV-2 protease resulted in the identification of four covalent inhibitors targeting the catalytic cysteine residue and five non-covalent inhibitors for which the binding was further investigated by molecular dynamics (MD) simulations. Predictive ADMET calculations on the nine compounds suggest promising pharmacokinetic properties.

Molecular characterization of TNF-β and IFN-γ in yellowfin seabream (Acanthopagrus latus, Hottuyn, 1782) and their immune responses to density stress during transport

The inflammatory cytokines TNF-β and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-β and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-β contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-β has a close affinity with TNF-β of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-β and AlIFN-γ are widely expressed in various tissues. AlTNF-β is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-β was significantly elevated in the intestine of the high-density group, while AlTNF-β transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-β and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4–5 cm fry or 1200 per bag for 1–2 cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.

ENGINEERING THE ACTIVE SITE OF FORMATE DEHYDROGE-NASE FROM STAPHYLOCOCCUS AUREUS: INTRODUCTION TO THE STRUCTURE OF THE ADDITIONAL LOOP AND HISTIDINE RESIDUES

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs signi cantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5-3 times) speci c activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of threedimensional structures and the alignment of amino acid sequences, substitutions promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in KMNAD+; the value of kcat has not changed. When Т250Н is replaced, an increase in KMNAD+ is observed, kcat decreases from 20 to 13 s-1. The replacement of K368H led to a slight increase in KMNAD+, kcat decreased from 20 s-1 to 6 s-1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in KMNAD+ and KMHCOO-. A bigger effect was observed for KMNAD+ - the difference was more than 10 times. For mutant SauFDH with insertions kcat signi cantly reduced to 4 s-1. Similar results were observed for mutants with multipoint substitutions. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH.

Identification of 1,3,6,8-Tetrahydroxynaphthalene Synthase (ThnA) from Nocardia sp. CS682.

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys138, Phe188, His270, and Asn303 as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividans TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividans TK24 and S. lividans pIBR25-thnA and found that thnA was only transcribed in the mutant.

Viral Protein 1 (VP1) Sequence-Based Genetic Diversity of SAT 2 FMDV Circulating in Ethiopia from 1990 to 2015.

Pathogen molecular epidemiology determines the origin of specific outbreaks locality of foot-and-mouth disease virus serotype South African Territories-2 sequence-based analysis of highly variable Viral Protein 1 (VP1), which helps to identify the evolution of this virus through time and space. The objective of this study was to compare the differences between SAT-2 VP1 sequences of FMDV circulated in Ethiopia from 1990 to 2015 at the genetic level. The nucleotide and amino acid sequences were analyzed using Basic Local Alignment Search Tools (BLAST), Multiple sequence alignment and sequence editing and Phylogenetic tree reconstruction. The nucleotide and amino acid sequences alignment, distance matrix, and phylogenetic tree constructions were done using the MEGA 6.0 software package. In this analysis, we found 76% nucleotide identities and amino acid similarities among the sequences. The overall group mean distance at nucleotide level was 19% with a mean intra-population diversity of 2%. The lowest sequence variation was observed among sequences obtained from the years 2007/09/10, 2014/15, and 1990/91 which was less than 5% among them. This analysis revealed that in the last 25 years, four different topotypes of the FMDV SAT-2 were circulating in Ethiopia. The Arg-Gly-Asp (RGD) amino acid (AA) motif at AA position 144-146 within the G-H loop of the VP1 protein of FMDV is conserved, but up- and downstream hyper-variable AA sequences are identified. In this study, it was observed that four topotypes (IV, XIV, XIII, and VII) were circulating in Ethiopia for 25 years. Further, compared with sequences from neighboring countries (Sudan, Kenya) confirmed the presence of these topotypes. Pertinent to this genetic diversity control strategies in Ethiopia should be based on having regular antigenic and genetic vaccine matching tests with the circulating strain within a defined period, space, transboundary nature of the disease and applying biosecurity measures.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

Creating high-resistant starch rice by simultaneous editing of SS3a and SS3b.

Gp120 envelope glycoproteins of HIV-1 Group M Subtype A and Subtype B differentially affect gene expression in human vascular endothelial cells

Abstract While HIV-1 Group M Subtype B is the most prevalent subtype in western countries, the majority of HIV-1 infections in eastern Africa and former Soviet Union countries are caused by Subtype A. However, most of the research on HIV has focused on Subtype B and information on the molecular mechanisms of Subtype A is virtually non-existent. The lack of such knowledge results in health disparities for the development of therapeutic strategies to prevent/treat HIV-associated complications. The present study examined envelope glycoprotein (gp120) of HIV-1 Group M Subtypes A and B. The amino acid sequence alignment analysis determined that gp120 proteins of A and B subtypes are 76% identical and share 86% similarity. The treatment of human vascular endothelial cells with the two gp120 proteins exhibited differential gene expression changes as determined by Proteome Profiler Arrays. Subtype A more potently downregulated prostasin, matrix metalloproteinase-2 (MMP-2), and receptor tyrosine-protein kinase erbB-3/Her3 than did Subtype B, while Subtype B was more effective in downregulating monocyte chemoattractant proteins (MCP-2 and MCP-3). Further, epidermal growth factor (EGF), a mediator of vascular complications, was found to be upregulated by gp120 of Subtype A, but not Subtype B. This is the first report of gp120 proteins affecting human host cells in an HIV subtype-specific manner, opening up the possibility that complications may occur differently in HIV patients throughout the world. Supported by grants from NIH (R21 AG73919, R03 AG71596)

Selection and Molecular Response of AHL-lactonase (aiiA) Producing Bacillus sp. Under Penicillin G-induced Conditions.

Quorum sensing (QS) is the process by which microorganisms employ chemicals called autoinducers (AIs) to communicate with their population. The QS mechanism generally controls the expression of the virulence related genes in bacteria. N-acyl homoserine lactones (AHLs) are the most widespread QS molecules. Due to their diverse AHL-lactonase activities, Bacillus species make particularly suitable candidates for procedures such as demolition of pathogenic bacterial QS signals and bioremediation of β-lactam antibiotics from contaminated environments. In this study, seven Bacillus strains with Quorum quenching (QQ) activity were isolated using an enrichment medium supplemented with Penicillin G (PenG). The AHL-lactonase encoding gene (aiiA) was amplified by PCR and sequenced. Amino acid sequences underwent multiple sequence alignment. Docking studies were carried out with both C6HSL and PenG ligand using AutoDock tools. The aiiA amino acid sequences of the isolates were found to be well conserved. Furthermore, amino acid sequence alignment revealed that 74.9% of amino acid sequences were conserved in the genus Bacillus. Docking of the C6HSL to wild type (3DHA) and H97D variant reduced the docking score by only 0.1kcal/mol for the mutated protein. When PenG docked with a higher (1.5kcal/mol) score as a ligand to wild-type and mutant receptors, the docking score for the mutated protein likewise decreased by 0.1kcal/mol. This research contributed to the diversification of organisms with QQ activity and beta-lactam antibiotic resistance. It also clarified the binding score of the PenG ligand to the Bacillus AHL lactonase molecule for the first time.

Functional Characterisation of Highly Conserved and Structurally Prominent Residues of 2-Methylisoborneol Synthase.

2-Methylisoborneol is a widespread musty odourant that is produced by many bacteria including actinomycetes, cyanobacteria and myxobacteria. Two 2-methylisoborneol synthases (MIBS) that are phylogenetically distant to the known enzyme from Streptomyces coelicolor were found to be highly active for 2-methylisoborneol biosynthesis. Based on the enzyme structure and on an amino acid sequence alignment, the MIBS from S. coelicolor was extensively studied through site-directed mutagenesis.

Combining Phage Display Technology with In Silico-Designed Epitope Vaccine to Elicit Robust Antibody Responses against Emerging Pathogen Tilapia Lake Virus.

Antigen epitope identification is a critical step in the vaccine development process and is a momentous cornerstone for the development of safe and efficient epitope vaccines. In particular, vaccine design is difficult when the function of the protein encoded by the pathogen is unknown. The genome of Tilapia lake virus (TiLV), an emerging virus from fish, encodes protein functions that have not been elucidated, resulting in a lag and uncertainty in vaccine development. Here, we propose a feasible strategy for emerging viral disease epitope vaccine development using TiLV. We determined the targets of specific antibodies in serum from a TiLV survivor by panning a Ph.D.-12 phage library, and we identified a mimotope, TYTTRMHITLPI, referred to as Pep3, which provided protection against TiLV after prime-boost vaccination; its immune protection rate was 57.6%. Based on amino acid sequence alignment and structure analysis of the target protein from TiLV, we further identified a protective antigenic site (399TYTTRNEDFLPT410) which is located on TiLV segment 1 (S1). The epitope vaccine with keyhole limpet hemocyanin (KLH-S1399-410) corresponding to the mimotope induced the tilapia to produce a durable and effective antibody response after immunization, and the antibody depletion test confirmed that the specific antibody against S1399-410 was necessary to neutralize TiLV. Surprisingly, the challenge studies in tilapia demonstrated that the epitope vaccine elicited a robust protective response against TiLV challenge, and the survival rate reached 81.8%. In conclusion, this study revealed a concept for screening antigen epitopes of emerging viral diseases, providing promising approaches for development and evaluation of protective epitope vaccines against viral diseases. IMPORTANCE Antigen epitope determination is an important cornerstone for developing efficient vaccines. In this study, we attempted to explore a novel approach for epitope discovery of TiLV, which is a new virus in fish. We investigated the immunogenicity and protective efficacy of all antigenic sites (mimotopes) identified in serum of primary TiLV survivors by using a Ph.D.-12 phage library. We also recognized and identified the natural epitope of TiLV by bioinformatics, evaluated the immunogenicity and protective effect of this antigenic site by immunization, and revealed 2 amino acid residues that play important roles in this epitope. Both Pep3 and S1399-410 (a natural epitope identified by Pep3) elicited antibody titers in tilapia, but S1399-410 was more prominent. Antibody depletion studies showed that anti-S1399-410-specific antibodies were essential for neutralizing TiLV. Our study demonstrated a model for combining experimental and computational screens to identify antigen epitopes, which is attractive for epitope-based vaccine development.