Amino Acid Sequence Alignment Research Articles

Molecular characterization of polyphenol oxidase between small and large leaf tea cultivars

Tea is a widely consumed beverage prepared using the fresh leaves of Camellia sinensis L. Tea plants are classified into small- and large-leaf varieties. Polyphenol oxidase (PPO), a crucial enzyme in tea manufacturing, catalyzes essential phenolic metabolites into different derivatives. To compare the molecular characteristics of CsPPO between cultivars, we cloned the full-length sequence of CsPPO cDNA from four representative tea cultivars in Taiwan. Amino acid sequence alignment analyses indicated that CsPPO is highly conserved. PPO exhibited similar enzymatic activity in different tea cultivars. Quantitative real-time polymerase chain reaction revealed no significant differences in the CsPPO transcript level between the small- and large-leaf varieties. However, tea harvested in summer and from low-altitude areas had a higher CsPPO transcript level than that harvested in winter and from high-altitude areas. Regulation of CsPPO by temperature was more significant in the small-leaf variety than in the large-leaf variety. The content of flavonoids and the expression of chalcone synthase, anthocyanidin synthase, and anthocyanidin reductase genes involved in the tea flavonoid biosynthesis pathway were higher in the large-leaf than in the small-leaf varieties, suggesting that the large-leaf variety had a higher antioxidative capacity than did the small-leaf variety. Our study compared the molecular properties of CsPPO between two tea varieties and clarified the physiological role of PPO in tea.

SaRCC1, a Regulator of Chromosome Condensation 1 (RCC1) Family Protein Gene from Spartina alterniflora, Negatively Regulates Salinity Stress Tolerance in Transgenic Arabidopsis.

A regulator of chromosome condensation 1 (RCC1) family protein has been functionally characterized to be involved in various cellular processes. In this study, one RCC1 gene named SaRCC1 was cloned from the full-length cDNA library of Spartina alterniflora. The open reading frame (ORF) of SaRCC1 was 1440 bp, and it encoded 479 amino acids with a calculated molecular mass of 51.65 kDa. Multiple amino acid sequence alignments showed that SaRCC1 had high identity with other plant RCC1s, and the phylogenetic analysis indicated that SaRCC1 had a closer affinity to Zea mays RCC1 family protein (ZmRCC1). SaRCC1 gene was induced under salt stress conditions, and its encoded protein was located in peroxisome. In order to further investigate the function of SaRCC1, transgenic Arabidopsis plants ectopically both sense-overexpressing and antisense-overexpressing SaRCC1 were generated. SaRCC1-overexpressing lines exhibited an increased salt and ABA hypersensitivity and reduced resistance to salinity stress. On the other hand, the transcripts of some stress-responsive genes in the SaRCC1 transgenic plants were affected in response to salinity stress. Our results provide evidence for the involvement of SaRCC1, negatively regulating salt stress responses by affecting stress-related gene expression in Arabidopsis.

Molecular characterization of carnivore protoparvovirus 1 circulating in domestic carnivores in Egypt.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV), now included in the unique species Carnivore protoparvovirus 1 (CPPV1), have been circulating in dogs and cats for several decades and are considered the causes of clinically important diseases, especially in young animals. While genetic evidence of the circulation of parvoviruses in Egyptian domestic carnivores has been provided since 2016, to date, all available data are based on partial fragments of the VP2 gene. This study reports the molecular characterization of CPPV strains from Egypt based on the full VP2 gene. Overall, 196 blood samples were collected from dogs and cats presented at veterinary clinics for routine medical assessment in 2019 in Egypt. DNA extracts were screened and characterized by real-time PCR. Positive samples were amplified by conventional PCR and then were sequenced. Nucleotide and amino acid changes in the sequences were investigated and phylogeny was inferred. Carnivore protoparvovirus DNA was detected in 18 out of 96 dogs (18.8%) and 7 of 100 cats (7%). Phylogenetic analyses based on the full VP2 gene revealed that 9 sequenced strains clustered with different CPV clades (5 with 2c, 2 with 2a, 1 with 2b, and 1 with 2) and 1 strain with the FPV clade. All three CPV variants were detected in dog and cat populations with a predominance of CPV-2c strains (7 of 18, 38.9%) in dog samples, thus mirroring the circulation of this variant in African, European, and Asian countries. Deduced amino acid sequence alignment revealed the presence of the previously unreported unique mutations S542L, H543Q, Q549H, and N557T in the Egyptian CPV-2c strains.

Stability is essential for insecticidal activity of Vip3Aa toxin against Spodoptera exigua

Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.

Molecular genotypic analysis of porcine circovirus type 2 reveals the predominance of PCV2d in Vietnam (2018-2020) and the association between PCV2h, the recombinant forms, and Vietnamese vaccines.

We conducted nucleotide and amino acid sequence alignment and phylogenetic analysis of porcine circovirus ORF2 (Cap protein) from 17 PCV2-positive clinical samples from nine different northern Vietnamese provinces (Mar 2018-Nov 2020), four local vaccines, and 77 reference strains. We identified one PCV2a (1/17 = 5.9%), five PCV2b (5/17 = 29.9%), and 11 PCV2d (11/17 = 64.7%) isolates, while only PCV2d was detected in 2020. Timeline analysis indicated an increasing predominance of PCV2d nationwide (2018-2020). With strong nodal support (98% for nucleotides and 74% for amino acids), the phylogenetic tree topology revealed a distinct PCV2h clade including recombinant/intermediate strains and local vaccines. The Cap protein sequences from 11 PCV2d field strains had the 2d-genotype-typical motif 86SNPLSV91 in loop CD, the motif TGID in loop GH-HI, and the motif 230PLNPK234 in loop CT. The PCV2h isolates (and vaccines) had the 86SNPLSV91, SAID, and 230L(N/H)PK234 motifs. Selection pressure analysis indicated positive selection at seven sites: A68N in immunoreactive region (IRR)-A; 119G and 130V in IRR-B; and 167L, T190(A/S), 194D and 202F in IRR-C. We identified PCV2h as the genotype of the recombinant strains, which resulted from intergenotype recombination of PCV2a, PCV2b, and PCV2d. The current data provide new information about the diversity, distribution, and dominance of the PCV2 genotype in Vietnam.

Overexpression of ZmEREBP60 enhances drought tolerance in maize

Apetala2/ethylene response factor (AP2/ERF) family of transcription factors plays important roles in plant development and stress responses. However, few members of this family have been functionally and mechanistically characterised in maize. In this study, we characterised a member of the AP2/ERF transcription factor family, ZmEREBP60 from maize. Amino acid sequence alignment and phylogenetic analysis showed that ZmEREBP60 belongs to cluster I of the AP2/ERF family. qRT-PCR analysis indicated that ZmEREBP60 expression was highly induced by drought in the roots, coleoptiles, and leaves. Subcellular localisation analysis revealed that ZmEREBP60 was localised in the nucleus. Moreover, overexpression of ZmEREBP60 enhanced tolerance to drought stress while alleviating the drought-induced increase in H2O2 accumulation and malondialdehyde content in transgenic lines. Transcriptome analysis showed that ZmEREBP60 regulates the expression of genes involved in H2O2 catabolism, water deprivation response, and abscisic acid signalling pathway. Collectively, as a new member of the AP2/ERF transcription factor family in maize, ZmEREBP60 is a positive regulator of plant drought response.

Detection and Molecular Characterization of Porcine Parvovirus 7 in Eastern Inner Mongolia Autonomous Region, China.

Porcine parvoviruses (PPV) and porcine circoviruses type 2 (PCV2) are widespread in the pig population. Recently, it was suggested that PPV7 may stimulate PCV2 and PCV3 replication. The present study aimed to make detection and molecular characterization of PPV7 for the first time in eastern Inner Mongolia Autonomous Region, China. Twenty-seven of ninety-four samples (28.72%) and five in eight pig farms were PPV7 positive. Further detection showed that the co-infection rate of PPV7 and PCV2 was 20.21% (19/94), and 9.59% (9/94) for PPV7 and PCV3. In addition, the positive rate of PPV7 in PCV2 positive samples was higher than that in PCV2 negative samples, supporting that PCV2 could act as a co-factor for PPV7 infection. In total, four PPV7 strains were sequenced and designated as NM-14, NM-19, NM-4, and NM-40. The amplified genome sequence of NM-14 and NM-40 were 3,999nt in length, while NM-19 and NM-4 were 3,996nt with a three nucleotides deletion at 3,097–3,099, resulting in an amino acid deletion in the Cap protein. Phylogenetic analysis based on the capsid amino acid (aa) sequences showed that 52 PPV7 strains were divided into two clades, and the four PPV7 strains in this study were all clustered in clade 1. The genome and capsid amino acid sequence of the four PPV7 strains identified in this study shared 80.0–96.9% and 85.9–100% similarity with that of 48 PPV7 reference strains selected in NCBI. Simplot analysis revealed that NM-19 and NM-4 strains were probably produced by recombination of two PPV7 strains from China. The amino acid sequence alignment analysis of capsid revealed that the four PPV7 strains detected in Inner Mongolia had multiple amino acid mutations in the 6 B cell linear epitopes compared with the reference strains, suggesting that the four PPV7 strains may have different characteristics in receptor binding and immunogenicity. In summary, this paper reported the PPV7 infection and molecular characterization in the eastern of Inner Mongolia Autonomous Region for the first time, which is helpful to understand the molecular epidemic characteristics of PPV7.

Genomic Comparisons of Alphacoronaviruses and Betacoronaviruses from Korean Bats.

Coronaviruses are well known as a diverse family of viruses that affect a wide range of hosts. Since the outbreak of severe acute respiratory syndrome, a variety of bat-associated coronaviruses have been identified in many countries. However, they do not represent all the specific geographic locations of their hosts. In this study, full-length genomes representing newly identified bat coronaviruses in South Korea were obtained using an RNA sequencing approach. The analysis, based on genome structure, conserved replicase domains, spike gene, and nucleocapsid genes revealed that bat Alphacoronaviruses are from three different viral species. Among them, the newly identified B20-97 strain may represent a new putative species, closely related to PEDV. In addition, the newly-identified MERS-related coronavirus exhibited shared genomic nucleotide identities of less than 76.4% with other Merbecoviruses. Recombination analysis and multiple alignments of spike and RBD amino acid sequences suggested that this strain underwent recombination events and could possibly use hDPP4 molecules as its receptor. The bat SARS-related CoV B20-50 is unlikely to be able to use hACE2 as its receptor and lack of an open reading frame in ORF8 gene region. Our results illustrate the diversity of coronaviruses in Korean bats and their evolutionary relationships. The evolution of the bat coronaviruses related ORF8 accessory gene is also discussed.

Genetic Diversity and Population Genetic Analysis of Plasmodium falciparum Thrombospondin Related Anonymous Protein (TRAP) in Clinical Samples from Saudi Arabia

The thrombospondin related anonymous protein (TRAP) is considered one of the most important pre-erythrocytic vaccine targets. Earlier population genetic studies revealed the TRAP gene to be under strong balancing natural selection. This study is the first attempt to analyze genetic diversity, natural selection, phylogeography and population structure in 199 clinical samples from Saudi Arabia using the full-length PfTRAP gene. We found the rate of nonsynonymous substitutions to be significantly higher than that of synonymous substitutions in the clinical samples, indicating a strong positive or diversifying selection for the full-length gene and the Von Willebrand factor (VWF). The nucleotide diversity was found to be π~0.00789 for the full-length gene; however, higher nucleotide diversity was observed for the VWF compared to the thrombospondin repeat region (TSP). Deduction of the amino acid sequence alignment of the PNP repeat region in the Saudi samples revealed six genotypes characterized by tripeptide repeat motifs (PNP, ANP, ENP and SNP). Haplotype network, population structure and population differentiation analyses indicated four distinct sub-populations in spite of the low geographical distance between the sampling sites. Our results suggest the likeliness of independent parasite evolution, creating opportunities for further adaptation, including host transition, and making malaria control even more challenging.

On the reliability and the limits of inference of amino acid sequence alignments.

MotivationAlignments are correspondences between sequences. How reliable are alignments of amino acid sequences of proteins, and what inferences about protein relationships can be drawn? Using techniques not previously applied to these questions, by weighting every possible sequence alignment by its posterior probability we derive a formal mathematical expectation, and develop an efficient algorithm for computation of the distance between alternative alignments allowing quantitative comparisons of sequence-based alignments with corresponding reference structure alignments.ResultsBy analyzing the sequences and structures of 1 million protein domain pairs, we report the variation of the expected distance between sequence-based and structure-based alignments, as a function of (Markov time of) sequence divergence. Our results clearly demarcate the ‘daylight’, ‘twilight’ and ‘midnight’ zones for interpreting residue–residue correspondences from sequence information alone.Supplementary information Supplementary data are available at Bioinformatics online.

β-arrestin interacts with TRAF6 to negatively regulate the NF-κB pathway in triangle sail mussel Hyriopsis cumingii

As members of arrestins family, β-arrestins are widely expressed in monocytes, macrophages, neutrophils and other immune cells. They can regulate the immune response of bodies through various ways. In the present study, a β-arrestin homolog named Hcβ-arrestin was cloned and identified from Hyriopsis cumingii. Predicted Hcβ-arrestin protein contained a conserved arrestin domain, which could be further divided into arrestin-N (39-192aa) and arrestin-C (211-365aa). Amino acid sequence alignment showed that it had the highest identity with Mytilus galloprovincialis and Mytilus edulis counterpart, which was 89.02% and 87.68%, respectively. Furthermore, real-time quantitative PCR analysis showed that the Hcβ-arrestin gene was widely expressed in the detected tissues and with the highest expression in hepatopancreas. The transcription of Hcβ-arrestin in hepatopancreas and gill of mussels was significantly up-regulated after stimulation with peptidoglycan, lipopolysaccharide (LPS) and polyinosinic polycytidylic acid. Knockdown of Hcβ-arrestin gene significantly increased the expression of some antibacterial effector genes, such as lysozyme, LPS-binding protein/bactericidal permeability increasing protein and theromacin in hepatopancreas and gills of LPS stimulated mussels, but only had little effect on TLR pathway genes. In addition, GST pull-down assay confirmed that Hcβ-arrestin can bind to HcTRAF6 protein in vitro. Dual luciferase reporter assay showed that the co-expression of HcTRAF6 and Hcβ-arrestin inhibited the activation of NF-κB reporter by HcTRAF6. These findings indicated that Hcβ-arrestins could interact with HcTRAF6 to negatively regulate the NF-κB pathway in H. cumingii.

Genome-wide identification and characterization of glutathione S-transferase gene family in Musa acuminata L. AAA group and gaining an insight to their role in banana fruit development

Glutathione S-transferases are a multifunctional protein superfamily that is involved in diverse plant functions such as defense mechanisms, signaling, stress response, secondary metabolism, and plant growth and development. Although the banana whole-genome sequence is available, the distribution of GST genes on banana chromosomes, their subcellular localization, gene structure, their evolutionary relation with each other, conserved motifs, and their roles in banana are still unknown. A total of 62 full-length GST genes with the canonical thioredoxin fold have been identified belonging to nine GST classes, namely tau, phi, theta, zeta, lambda, DHAR, EF1G, GHR, and TCHQD. The 62 GST genes were distributed into 11 banana chromosomes. All the MaGSTs were majorly localized in the cytoplasm. Gene architecture showed the conservation of exon numbers in individual GST classes. Multiple Em for Motif Elicitation analyses revealed few class-specific motifs and many motifs were found in all the GST classes. Multiple sequence alignment of banana GST amino acid sequences with rice, Arabidopsis, and soybean sequences revealed the Ser and Cys as conserved catalytic residues. Gene duplication analyses showed the tandem duplication as a driving force for GST gene family expansion in banana. Cis-regulatory element analysis showed the dominance of light-responsive element followed by stress- and hormone-responsive elements. Expression profiling analyses were also done by RNA-seq data. It was observed that MaGSTs are involved in various stages of fruit development. MaGSTU1 was highly upregulated. The comprehensive and organized studies of MaGST gene family provide groundwork for further functional analysis of MaGST genes in banana at molecular level and further for plant breeding approaches.

Flavorubredoxin, a Candidate Trigger Related to Thrombotic Thrombocytopenic Purpura: Screening of the Complete Genome of a Salmonella enterica Serovar Typhimurium Isolate From an AIDS Case

Thrombotic thrombocytopenic purpura (TTP) is one of the two classic thrombotic microangiopathy (TMA) diseases which could be induced by infections. To the best of our knowledge, this is the first report of an acquired immunodeficiency syndrome (AIDS) patient with acquired TTP induced by infection with Salmonella enterica serovar Typhimurium (hereafter, S. Typhimurium) isolate, S. Typhimurium_zhang, which was confirmed by serology and genetic taxonomy. The literature review identified 17 TMA-related genes encoding the candidate triggers, which were searched in the annotated genome sequence of S. Typhimurium_zhang. Anaerobic nitric oxide reductase flavorubredoxin (FlRd), encoded by norV which is related to another TMA, haemolytic uraemic syndrome (HUS), was found in S. Typhimurium_zhang. Basic local alignment search tool (BLAST) analysis revealed that norV and FlRd in S. Typhimurium_zhang, as well as eight S. Typhimurium type strains, have high identity with HUS-related Escherichia coli O157:H7 strain TW14359. Similar results were obtained from the BLAST analysis of 73 S. enterica isolates for congenital TTP which was also previously reported to be triggered by S. enterica. Phylogenetic analysis and amino acid sequence alignment revealed that FlRd was functional and highly conservative on 69 Enterobacteriaceae, including S. Typimurium_zhang and TW14359. In brief, we found norV in the genome of a S. Typhimurium clinical isolate that induced TTP in an AIDS patient. FlRd, the protein encoded by norV, probably triggered the TTP and was highly conservative, functional, and widespread in S. enterica and Enterobacteriaceae. More in vitro and in vivo studies are required to confirm our findings and determine the underlying mechanism.

Cloning and homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS) from Withania somnifera revealed alterations in metabolic flux towards gibberellic acid biosynthesis.

Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis.

Molecular characterization and expression analysis of IL-10 and IL-20L genes in swamp eel (Monopterus albus) against Aeromonas veronii infection

IL-10 cytokine family belongs to type Ⅱhelix cytokine, which has many common characteristics and different biological functions. In this paper, the IL-10 and IL-20L genes in the IL-10 family of swamp eel (Monopterus albus) were characterized for the first time, and their expression patterns after stimulation by Aeromonas veronii were studied. Through phylogenetic tree, amino acid sequence alignment and similarity analysis, eel IL-10 and IL-20 were the closest to their counterparts of tilapia, and fish IL-20L molecules were clustered into a branch alone, and then converged with IL-19/IL-20/IL-24 of higher vertebrates. There are two conserved motifs in eel IL-10 and IL-20L, and the motif in IL-10 is completely consistent with the conserved signature motif of IL-10 family. Eel IL-10 has 6 α helices and 4 conserved cysteines, and IL-20 has 5 α helices and 6 conserved cysteines, in which helix E′ is equivalent to helices E and F of IL-10. These results indicate that fish IL-10 and IL-20L cytokines were evolutionarily conservative. The gene structure of IL-10 and IL-20 of all selected species consists of five coding exons and four 0-phase introns, which are closely linked at the corresponding loci. Our study supports the hypothesis that the IL-20 subfamily cytokines share a common origin with IL-10. Whether it is tissue expression or induced expression, eel IL-10 has a higher expression level than IL-20L, but both of them may be involved in the late stage of the host's response to bacterial infection, and there are differences between them and other fish expression patterns. It is hoped that these research results will help to understand the immune system of fish and formulate disease prevention and control measures.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Investigating the Mechanistic Strategies of the Two‐component FMN‐dependent Alkanesulfonate Monooxygenase Systems

The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.

A human coronavirus OC43-derived polypeptide causes neuropathic pain.

Human coronaviruses have been recently implicated in neurological sequelae by insufficiently understood mechanisms. We here identify an amino acid sequence within the HCoV‐OC43 p65‐like protein homologous to the evolutionarily conserved motif of myelin basic protein (MBP). Because MBP‐derived peptide exposure in the sciatic nerve produces pronociceptive activity in female rodents, we examined whether a synthetic peptide derived from the homologous region of HCoV‐OC43 (OC43p) acts by molecular mimicry to promote neuropathic pain. OC43p, but not scrambled peptides, induces mechanical hypersensitivity in rats following intrasciatic injections. Transcriptome analyses of the corresponding spinal cords reveal upregulation of genes and signaling pathways with known nociception‐, immune‐, and cellular energy‐related activities. Affinity capture shows the association of OC43p with an Na+/K+‐transporting ATPase, providing a potential direct target and mechanistic insight into virus‐induced effects on energy homeostasis and the sensory neuraxis. We propose that HCoV‐OC43 polypeptides released during infection dysregulate normal nervous system functions through molecular mimicry of MBP, leading to mechanical hypersensitivity. Our findings might provide a new paradigm for virus‐induced neuropathic pain.

Heterologous Expression of LiSEP3 from Oriental Lilium Hybrid 'Sorbonne' Promotes the Flowering of Arabidopsis thaliana L.

The MADS-box gene family has multiple molecular and biological functions in plants. Here, the LiSEP3 gene of the MADS-box gene family of' 'Sorbonne' was obtained by homologous cloning using the petals of the flowering stage of Lilium Oriental Hybrid 'Sorbonne.' The ORF full-length sequence is 729bp, encoding 242 amino acids. Bioinformatics analysis showed that the relative molecular weight of the LiSEP3 protein is 27.67 kD and the isoelectric point (pI) is 9.16. The prediction result of the gene positioning is transcription in its nucleus. Homologous alignment of amino acid sequences showed that the protein not only had typical MADS-box and K-box domains, but also contained two short and relatively conservative SEP motifs. The phylogenetic tree showed that the amino acid sequence encoded by the LiSEP3 gene had the closest relationship with SEP3 in monocotyledon plants such as Apostasia odorata. The results of real-time PCR showed that LiSEP3 gene was mainly expressed in petal. During flower development, the expression level of the LiSEP3 gene showed an overall trend of initially increasing and then decreasing. The flowering time of LiSEP3 transgenic Arabidopsis thaliana L. plants was earlier than that of wild-type Arabidopsis thaliana L. plants, compared with wild type, the number of rosette leaves is less. In the transgenic plants, the expression of flowering-associated AtSPL5 and AtGI genes was up-regulated, while the expression of AtSVP and AtFRI genes that inhibit flowering was down-regulated, which was consistent with the statistical results of the flowering time of LiSEP3 transgenic plants. Our results illustrate that the heterologous expression of SEP3 functional genes in the MADS-box family promoted the flowering period of transgenic plants of this hybrid. This research provides a theoretical basis for improving the flowering period of ornamental plants through plant genetic engineering technology and enhancing their economic and social values.

Genetic diversity and evolution of goose astrovirus in the east of China.

Goose astrovirus (GAstV), an agent of fatal visceral gout in goslings, has been widely circulating in eastern China since 2017, but little is known about its genetic diversity and systematic evolution. In this study, we isolated and sequenced nine nearly full-length GAstV genomes and conducted comprehensive genetic diversity and evolutionary analysis and compared them with other reported GAstV sequences. Our results indicated that two genotypic species of GAstV were circulating in China, and GAstV-2 subgenotype II-c had arisen as the dominant genotype in Shandong province and across the whole country. Multiple alignments of GAstV amino acid sequences revealed several characteristic mutations in GAstV-2 II-c strains, as well as additional residues in the nine new isolates which varied over time. Phylogenetic analysis of three open reading frames demonstrated different evolutionary histories. Evidence of natural recombination was also detected in GAstV, with most of the recombination occurring in the GAstV-2 II-c subgenotype. Molecular adaptation analyses revealed that the evolution of GAstV was shaped by strong negative selection, although a number of amino acids, which potentially affect host infection and cell entry, were subjected to positive pressure. Overall, these findings improve our understanding of the epidemiology and evolution of GAstV and may help in the development of vaccines and diagnostics.