Amino Acid Sequence Alignment Research Articles

Complete genome analysis of a novel hypovirus in the phytopathogenic fungus Monilinia fructicola.

Monilinia fructicola is one of the most devastating fungal diseases of rosaceous fruit crops, both in the field and postharvest, causing significant yield losses. Here, we report the discovery of a novel positive single-stranded RNA virus, Monilinia fructicola hypovirus 3 (MfHV3), in a strain (hf-1) of the phytopathogenic fungus Monilinia fructicola. The complete genome of MfHV3 is 9259 nucleotides (nt) in length and contains a single large open reading frame (ORF) from nt position 462 to 8411. This ORF encodes a polyprotein with three conserved domains, namely UDP-glycosyltransferase, RNA-dependent RNA polymerase (RdRp), and DEAD-like helicase. The MfHV3 polyprotein shares the highest similarity with Colletotrichum camelliae hypovirus 1. Phylogenetic analysis indicated that MfHV3 clustered with members of the genus Betahypovirus within the family Hypoviridae. Taken together, the results of genomic organization comparisons, amino acid sequence alignments, and phylogenetic analysis convincingly show that MfHV3 is a new member of the genus Betahypovirus, family Hypoviridae.

Occurrence, distribution, and genetic diversity of faba bean viruses in China.

With worldwide cultivation, the faba bean (Vicia faba L.) stands as one of the most vital cool-season legume crops, serving as a major component of food security. China leads global faba bean production in terms of both total planting area and yield, with major production hubs in Yunnan, Sichuan, Jiangsu, and Gansu provinces. The faba bean viruses have caused serious yield losses in these production areas, but previous researches have not comprehensively investigated this issue. In this study, we collected 287 faba bean samples over three consecutive years from eight provinces/municipalities of China. We employed small RNA sequencing, RT-PCR, DNA sequencing, and phylogenetic analysis to detect the presence of viruses and examine their incidence, distribution, and genetic diversity. We identified a total of nine distinct viruses: bean yellow mosaic virus (BYMV, Potyvirus), milk vetch dwarf virus (MDV, Nanovirus), vicia cryptic virus (VCV, Alphapartitivirus), bean common mosaic virus (BCMV, Potyvirus), beet western yellows virus (BWYV, Polerovirus), broad bean wilt virus (BBWV, Fabavirus), soybean mosaic virus (SMV, Potyvirus), pea seed-borne mosaic virus (PSbMV, Potyvirus), and cucumber mosaic virus (CMV, Cucumovirus). BYMV was the predominant virus found during our sampling, followed by MDV and VCV. This study marks the first reported detection of BCMV in Chinese faba bean fields. Except for several isolates from Gansu and Yunnan provinces, our sequence analysis revealed that the majority of BYMV isolates contain highly conserved nucleotide sequences of coat protein (CP). Amino acid sequence alignment indicates that there is a conserved NAG motif at the N-terminal region of BYMV CP, which is considered important for aphid transmission. Our findings not only highlight the presence and diversity of pathogenic viruses in Chinese faba bean production, but also provide target pathogens for future antiviral resource screening and a basis for antiviral breeding.

PHYLOGENETIC CONNECTIONS AND COMPARISONS OF THE AMINO ACID SEQUENCES OF SEVERAL PEPTIDE INDUCERS OF MICROBIAL ORIGIN- A FOUNDATION STUDY FOR DEVELOPMENT OF NOVEL BIOPESTICIDES

Peptides of microbial origin stand out as one of theprominent tools to elicit plant immunity. These peptides comprise one of thekey strategies of integrated pest management and are considered as candidatesto develop novel biopesticides. Manyresearch investigations have proved their potential in fending off plantpathogens and were described as sustainable plant protection strategies. Thepresent study was attempted to discover phylogenetic relationships and compareamino acid sequence alignments of various peptide elicitors of microbialorigin. Phylogenetic analysis of 33 microbial peptide elicitors resulted in twoclusters, one cluster contained 19 flagellin sequences, which is furtherdivided into one major (15 peptide sequences) and one (4 peptide sequences)minor subclusters. Further amino acid sequence alignments were carried outbased on the evolutionary relationships among the peptides. The amino acidsequence alignment of flagellin sequences using Clustal W did not present conservativeamino acid sequences except Serine (S), Alanine (A) and Aspartic acid (D).These conserved amino acids (SAxD) that are positioned in the protruding loopmay play a vital role in recognition by plant surface receptors. Alignment ofamino acid sequences of cold shock protein, xylanase, elongation factor andharpin from various sources did not present conservative amino acid sequencesexcept glycine. These investigations lay a theoretical foundation for exploringmany more microbial peptides for inducing plant resistance.

Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQβ-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQβ eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Evaluation of the anti-trypanosoma activity of alkaloids from Pilocarpus microphyllus by molecular docking: A search for new drugs for Chagas disease

Trypanosoma cruzi and Trypanosoma brucei, respectively, transmit the neglected tropical diseases (NTDs) Chagas disease and sleeping sickness. They cause symptoms that are harmful to public health. Treatment often has adverse effects, including high toxicity and neurosensory disorders. Studies aimed at developing pharmacological technologies are essential. In this study, we performed computational simulations based on in silico predictions. The amino acid sequence of the proteins for both parasites, T. cruzi and T. brucei, and their three-dimensional (3D) structures were obtained and drawn using the UniProt and Swiss model servers, respectively; later, they were used for in silico molecular docking studies. The GaussView software was used to get the two-dimensional (2D) and three-dimensional (3D) ligands of epiisopiloturine, epiisopilosine, pilosine, isopilosine, macaubine, and pilocarpine. To perform the optimization and frequency calculations of the alkaloids, the Gaussian 09w software and the Density Functional Theory (DFT) method were used with the hybrid functional B3LYP and the STO-3G support base. All the output files generated after the calculations were used to create the input files for the bond energy calculations. For the molecular docking studies, we have evaluated the side-ligands protein interactions and antiparasitic activity of the ligands. The anti-Trypanosomatidae potential of alkaloids extracted from Pilocarpus microphyllus “jaborandi” were evaluated by molecular docking. The results of the interactions of the 6 alkaloids: isopilosine, epiisopiloturine, epiisopilosine, pilocarpine, macubine and pilosine, with the proteins trypanothione reductase, sterol 14-alpha demethylase, phosphoinositide phospholipase c, farnesyl pyrophosphate synthase, nitroreductase, from the parasites T. cruzi and T. brucei, demonstrated that for 5 of them, the simulations showed minimums energy to be characterized as reactions that occur in biological systems spontaneously. For trypanothione reductase-epiisopilosine for both parasites, the alkaloid was observed to bind to the amino acid Glu467 through hydrogen bonds. For phosphoinositide phospholipase C-epiisopilosine, parameter values found were -8.2 Kcal.mol-1, only the amino acid Asn262 had interaction through hydrogen bonding. Other hydrophobic interactions were also observed between the proteins and epiisopilosine. Regarding the results of the alignment of amino acid sequences for both parasites, it was found a similarity of 83.30% and 52.38% between the nucleotide sequences of T. cruzi and T. brucei for trypanothione reductase and phosphoinositide phospholipase C, concluding that the best candidate for therapy against Chagas disease and sleeping sickness would be epiisopilosine alkaloid.

Molecular characterisation of a novel sadwavirus infecting cattleya orchids in Australia

The complete genome sequence of a novel sadwavirus infecting cattleya orchids in South East Queensland is described. Isometric virions of c. 27 nm diameter were observed in sap extracts viewed under a transmission electron microscope, and the genome sequence of this virus was determined by high-throughput sequencing. The viral genome consists of two RNA components, 5,910 and 4,435 nucleotides (nt) in length, each encoding a long polyprotein, with predicted cleavage sites at H/Y, E/G, Q/S, and Q/G for the RNA1 and T/G for the RNA2 translation products, respectively. RNA2 has an additional small ORF of 684 nt near the 3ʹ untranslated region. Phylogenetic analysis based on an amino acid sequence alignment of the Pro-Pol region suggested that this virus is most closely related to pineapple secovirus A, a member of the subgenus Cholivirus, but warrants classification as a member of a new species because it exhibited no more than 64% amino acid identity in pairwise sequence comparisons. Because of the prominent purple ringspots that were observed on the leaves of some of the plants, we propose the name “cattleya purple ringspot virus” for this virus (suggested species name: “Sadwavirus cattleyacola”).

Assessment of pathogenicity and functional characterization of APPL1 gene mutations in diabetic patients.

Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) plays a crucial role in regulating insulin signaling and glucose metabolism. Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14 (MODY14). Currently, only two mutations [c.1655T>A (p.Leu552*) and c.281G>A p.(Asp94Asn)] have been identified in association with this disease. Given the limited understanding of MODY14, it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations. To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain. Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study. Whole exome sequencing was performed on the patients as well as their family members. The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis. In addition, the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments. Finally, the impact of these variants on APPL1 protein expression and the insulin pathway were assessed, and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored. A total of five novel mutations were identified, including four missense mutations (Asp632Tyr, Arg633His, Arg532Gln, and Ile642Met) and one intronic mutation (1153-16A>T). Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions. The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster. In addition, multiple alignment of amino acid sequences showed that the Arg532Gln, Asp632Tyr, and Arg633His variants were conserved across different species. Moreover, in in vitro functional experiments, both the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels, indicating their pathogenic nature. Therefore, based on the patient's clinical and family history, combined with the results from bioinformatics analysis and functional experiment, the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were classified as pathogenic mutations. Importantly, all these mutations were located within the phosphotyrosine-binding domain of APPL1, which plays a critical role in the insulin sensitization effect. This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.

Potential SLA Hp-4.0 haplotype-restricted CTL epitopes identified from the membrane protein of PRRSV induce cell immune responses.

Swine leukocyte antigen (SLA) class I molecule-restricted T-cell epitopes, which induce cytotoxic T lymphocyte (CTL) responses, play a critical role in the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) and the development of efficient protective vaccines. The SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01 alleles, assigned the Hp-4.0 haplotype, are highly prevalent and usually present in all pig breeds. However, the SLA Hp-4.0 haplotype-restricted CTL epitopes in the structural membrane (M) protein of PRRSV are still unknown. In this study, we predicted 27 possible 9-mer epitope peptides in M protein with high binding scores for SLA-1*04:01:01 using CTL epitope prediction tools. In total, 45 SLA class I complexes, comprising the predicted peptide, extracellular region of the SLA-I molecules, and β2-microglobulin, were constructed in vitro to detect the specific binding of these peptides to SLA-1*04:01:01 (27 complexes), SLA-2*04:01 (9 complexes), and SLA-3*04:01 (9 complexes), respectively. Our results showed that the M27 (T91WKFITSRC), M39 (N130HAFVVRRP), and M49 (G158RKAVKQGV) peptides bind specifically to SLA-1*04:01:01, SLA-2*04:01, and SLA-3*04:01, respectively. Subsequently, using peripheral blood mononuclear cells (PBMCs) isolated from the homozygous Hp-4.0 and Hp-26.0 haplotype piglets vaccinated with commercial PRRSV HuN4-F112 strain, we determined the capacities of these 27 potential peptides to stimulate their proliferation with a Cell Counting Kit-8 and their secretion and expression of interferon gamma (IFN-γ) with an ELISpot assay and real-time qPCR, respectively. The immunological activities of M27, M39, and M49 were therefore confirmed when they efficiently induced PBMC proliferation and IFN-γ secretion in PBMCs from piglets with the prevalent SLA Hp-4.0 haplotype. The amino acid sequence alignment revealed that M27, M39, and M49 are highly conserved among 248 genotype II PRRSV strains collected between 1998 and 2019. These findings contribute to the understanding of the mechanisms of cell-mediated immune responses to PRRSV. Our study also provides a novel strategy for identifying and confirming potential SLA haplotype-restricted CTL epitopes that could be used to develop novel peptide-based vaccines against swine diseases.

Isolation and complete genomic characterization of a Movar 33/63-like Japanese bovine herpesvirus 4 from a calf with respiratory disease.

Bovine herpesvirus 4 (BoHV-4) is an indigenous virus in cattle prevalent mainly in North and South American countries and European countries, but the genomic sequences and genetic characteristics of Japanese strains have not been reported. BoHV-4 is suspected, but not proven, to be associated with various diseases. In the present study, we isolated BoHV-4 from a 10-month-old Japanese Black calf with respiratory symptoms in Japan. To identify the genetic characteristics of the isolate named strain SG20, complete genome sequencing was performed using a combination of next-generation and Sanger sequencing technologies. The complete long unique coding region (LUR) of SG20 was found to comprise 108,819 nucleotides with 41.4% GC content and contain at least 78 open reading frames. It shares 83.4 to 99.3% overall nucleotide identity with six BoHV-4 strains available in the database. The deduced amino acid sequence alignment revealed that SG20 contains genotype 1-specific features of BoHV-4, such as amino acid substitutions and insertions within the glycoprotein B region. Phylogenetic analyzes based on the nucleotide sequences of ORF20 indicated that the virus belonged to genotype 1 (Movar 33/63-like group). The strain was also analyzed using the complete LUR and placed in the same clade as a strain recently isolated from China, but it was distinct from American and European BoHV-4 strains of genotype 1. Although further genomic and epidemiologic information is needed, our results help elucidate the molecular epidemiology of BoHV-4 and provide a foundation for future studies.

Microbe Profile: The Lactobacillaceae.

Graphical abstract Phylogeny, host association and genome size in the Lactobacillaceae . The phylogenetic tree of 287 unique species and 15 strains with unassigned species was built using concatenated alignment of amino acid sequences of core genes, produced by SCARAP (https://journals.asm.org/doi/10.1128/msystems.00264-19) as an input into IQ-TREE2 (https://academic.oup.com/mbe/article/37/5/1530/5721363) with model parameters are described in []. Coloured branches represent species divisions established in []. Coloured taxon labels indicate major clades and recently established genera. The solid circles by the species names represent genome sizes. The coloured bands on the outer ring indicate the typical habitat based on data in [].

Genome-wide analysis of respiratory burst oxidase homolog gene family in pea (Pisum sativum L.).

Plant respiratory burst oxidase homologs (RBOHs) are key enzymes regulating superoxide production, which is important for plant development and responses to biotic and abiotic stresses. This study aimed to characterize the RBOH gene family in pea (Pisum sativum L.). Seven PsRBOH genes were identified in the pea genome and were phylogenetically clustered into five groups. Collinearity analyses of the RBOHs identified four pairs of orthologs between pea and soybean. The gene structure analysis showed that the number of exons ranged from 6 to 16. Amino acid sequence alignment, conserved domain, and conserved motif analyses showed that all seven PsRBOHs had typical features of plant RBOHs. The expression patterns of PsRBOH genes in different tissues provided suggested their roles in plant growth and organ development. In addition, the expression levels of PsRBOH genes under different abiotic stresses were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that PsRBOH genes exhibited unique stress-response characteristics, which allowed for functional diversity in response to different abiotic stresses. Furthermore, four PsRBOHs had a high probability of localization in the plasma membrane, and PsRBOH6 was localized to the plasma membrane and endoplasmic reticulum. The results of this study provide valuable information for further functional analysis of pea RBOH genes and their role in plant adaptation to climate-driven environmental constraints.

Equine influenza outbreak in Eastern of Algeria in 2021: The first introduction of Florida Clade 1 to Maghreb area

We have performed an equine influenza (EI) serological study of the equine population in Algeria by testing 298 serum samples collected between February and August 2021 from 5 provinces. The results were obtained performing an NP-ELISA. Our results revealed that 49.3% (147/298) samples positive for antibodies to EI (H3N8). During this study and after a gap of one decade an outbreak of EI was reported in Algeria in the first week of March 2021. The disease was confirmed by virus detection from the nasal swabs (n = 39) by qRT-PCR and by identifying 5 EI seroconversion. The virus sequences were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 1 of the Florida sublineage in the American lineage. This study indicate the first detection of FC1 strain of EIV in Maghreb area.

Bioinformatics Analysis of Global Diversity in Meningococcal Vaccine Antigens over the Past 10 Years: Vaccine Efficacy Prognosis.

Neisseria meningitidis (N. meningitidis) serogroup B (MenB) is the leading cause of invasive meningococcal disease worldwide. The pathogen has a wide range of virulence factors, which are potential vaccine components. Studying the genetic variability of antigens within a population, especially their long-term persistence, is necessary to develop new vaccines and predict the effectiveness of existing ones. The multicomponent 4CMenB vaccine (Bexsero), used since 2014, contains three major genome-derived recombinant proteins: factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA). Here, we assessed the prevalence and sequence variations of these vaccine antigens in a panel of 5667 meningococcal isolates collected worldwide over the past 10 years and deposited in the PubMLST database. Using multiple amino acid sequence alignments and Random Forest Classifier machine learning methods, we estimated the potential strain coverage of fHbp and NHBA vaccine variants (51 and about 25%, respectively); the NadA antigen sequence was found in only 18% of MenB genomes analyzed, but cross-reactive variants were present in less than 1% of isolates. Based on our findings, we proposed various strategies to improve the 4CMenB vaccine and broaden the coverage of N. meningitidis strains.

Histone acetylation is associated with pupal diapause in cotton bollworm, Helicoverpa armigera.

Diapause is an environmentally preprogrammed period of arrested development that is important to insect survival and population growth. Histone acetylation, an epigenetic modification, has several biological functions, but its role in agricultural pest diapause is unknown. In this study, we investigated the role of histone H3 acetylation in the diapause of Helicoverpa armigera. The histone H3 gene of H. armigera was cloned, and multiple sequence alignment of amino acids revealed that the potential lysine acetylation sites were highly conserved across species. Investigation of histone H3 acetylation levels in diapause- and nondiapause-type pupae showed that acetylation levels were down-regulated in diapause-type pupae and were lower in diapausing pupae compared to nondiapause pupae. By screening the genome, six histone acetyltransferase (HAT) and eight histone deacetylase (HDAC) genes responsible for antagonizing catalytic histone acetylation modifications were identified in H. armigera, and most of them exhibited different expression patterns between diapause- and nondiapause-type pupae. To elucidate the effect of histone H3 acetylation on diapause in H. armigera, the diapause pupae were injected with the histone acetylation activator trichostatin A (TSA). The results indicated that TSA injection increased the levels of histone H3 acetylation, causing the diapausing pupae to revert to development. Furthermore, transcriptome analysis revealed that 259 genes were affected by TSA injection, including genes associated with metabolism, resistance, and immunological responses. These results suggest that histone acetylation is inseparably related to the pupal diapause of H. armigera, which promises to be a potential target for pest control. © 2023 Society of Chemical Industry.

2608. Circulating Respiratory Syncytial Virus Genotypes in Hospitalized Children with Bronchiolitis in the United Arab Emirates

Abstract Background Respiratory syncytial virus (RSV) is a major cause of acute respiratory illness in young children and elderly people worldwide. It has a complex molecular epidemiology with multiple strains cocirculating during a single epidemic. Whole genome sequencing is a valuable tool for monitoring genetic diversity and providing genomic information essential for antiviral and vaccine development. In the United Arab Emirates (UAE), little is known about the RSV circulating genotypes, their evolution, and transmission dynamics. Using next-generation sequencing, this study aims to identify RSV genotypes circulating among young children with bronchiolitis in Al Ain City, UAE. Methods This cross-sectional study involved 100 children under two years of age who were hospitalized at a tertiary care facility between April and December 2021 with RSV-positive bronchiolitis. Nasopharyngeal samples were obtained, within 24 hours of hospitalization, for RSV genotyping. MinION nanopore sequencer was used to analyze the whole viral genome. We only sequenced samples with cycle threshold (Ct) < 30 (n=70). After sequencing, 7 samples were not analyzed due to long gaps and small lengths. The remaining 63 samples included 59 RSV-A and 4 RSV-B isolates. Results The median age of the studied children was 4.3 months, interquartile range (2.1-10.4), with 56% below the age of 6 months. There was no significant difference in the clinical characteristics of children having RSV-A compared to those infected with RSV-B. Phylogenetic analysis showed that out of the 59 RSV-A sequences, 58 clustered with the NA1 genotype, and 1 sequence clustered with the GA1 genotype. All RSV-B sequences were classified as BA2 genotypes. The amino acid sequence alignment of the G gene revealed that all 58 NA1 genotypes had 24 amino acid duplication regions from 247 to 282 amino acid positions. We also found four substitutional changes at L248I, H258Q, L274P, and P276Q. Conclusion Phylogenetic analysis revealed that the subtype A NA1 genotype was the dominant strain among the studied children in Al Ain, UAE during the 2021 season. As vaccine development advances, further sequencing of RSV is needed to better understand viral origin, transmission, and genetic recombination, particularly in under-studied populations. Disclosures All Authors: No reported disclosures

Expression dynamics indicate the involvement of SPG7 in the reproduction and spermiogenesis of Phascolosoma esculenta

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

Molecular cloning and heterologous expression analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase gene in Centella asiatica L.

Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.

Expression and characterization of an organic solvent tolerant recombinant lipase from Staphylococcus capitis SH6 for food wastewater treatment

The study illustrated here aims on an organic solvent tolerant lipase from Staphylococcus capitis (SCL). The gene part, encoding the mature lipase, was cloned and sequenced. The concluded polypeptide sequence, equivalent to the protein, consist of 388 amino acid residues with a molecular mass of about 45 kDa. A structure-based alignment of the SCL amino acid sequence shows high identities with those many staphylococcal lipases. From this alignment of sequences, the catalytic triad (Ser 117, Asp 308 and His 347) of SCL could be identified. The mature part of the SCL was expressed in Escherichia coli and the recombinant lipase (r-SCL) was purified to homogeneity. The purified r-SCL presented a quite interesting stability at low temperatures (< 30 °C) and the enzyme was found to be highly stable in polar organic solvent and at a pH ranging from 3 to 12. After that, we have demonstrated that the recombinant enzyme may be implicated in the biodegradability of oily wastewater from effluents of fast-food restaurants; the maximum conversion yield into fatty acids obtained at 30 °C, was 65%.

Immunobiological properties and structure analysis of group 13 allergen from Blomia tropicalis and its IgE-mediated cross-reactivity

Blomia tropicalis is an important species of allergenic mite. Structurally related cross-reactive allergens are involved in pathogenesis of clinical symptoms. The present study focused on recombinant allergen rBlo t 13 from B. tropicalis, including investigation of its structure, immunological properties, IgE-mediated cross-reactivity. In this work, the prokaryotic expression plasmids pET-28(a)-Blo t 13, pET-28(a)-Der f 13, and pET-28(a)-Tyr p 13 were constructed, transformed into E. coli Rosetta (DE3) pLysS, and purified by nickel affinity chromatography, respectively. By using ELISA, the IgE-binding rates were detected for rBlo t 13 and its epitope peptides, as well as the cross-reactivity among rBlo t 13, rDer f 13, and rTyr p 13. The tertiary structure of rBlo t 13 was resolved using X-ray diffraction at 2.0 Å resolution. Using IgE-ELISA, the IgE binding rate of rBlo t 13 was 60 % with Blomia tropicalis-positive sera. In the experiments of ELISA for cross-reactivity with rBlo t 13 on solid phase, the inhibition rates were 65 %, 57 % and 63 % for rBlo t 13, rDer f 13, and rTyr p 13, respectively. The structure of Blo t 13 protein contains a β-barrel structure which is composed of 10 β strands and has 2 α helices at the end of the barrel. Comparison of the tertiary structures of rBlo t 13, rDer f 13, and rTyr p 13 revealed that the β-barrel structure is highly conserved, consistent with the alignment of amino acid sequences. We obtained the recombinant protein rBlo t 13, demonstrated its cross-reactivity with Der f 13 and Tyr p 13 due to their structural similarity.

Molecular characterization and expression analysis of nine toll like receptor (TLR) genes in Scortum barcoo under Streptococcus agalactiae infection

Toll like receptors (TLRs) are important pattern recognition receptors participating in innate immune system. Up to now, no TLR has been identified in Jade perch (Scortum barcoo). In this study, we successfully identified 9 members of TLRs from the Jade perch. Amino acid sequence alignment analysis showed that the whole sequences of these TLRs were highly conserved among different fish species, especially in LRR, TM and TIR domains. Phylogenetic analysis revealed that each SbTLR was successfully grouped into corresponding gene family of teleosts. Expression analysis showed that most SbTLRs mainly expressed in liver, spleen, muscle and skin, while expressed less in brain and stomach. After Streptococcus agalactiae infection, expression of SbTLR2, SbTLR5S and SbTLR22 were significantly upregulated, while SbTLR3, SbTLR5M, SbTLR9, SbTLR13, and SbTLR14 were significantly downregulated. In all, this research first reported molecular characterization and expression profiles of 9 TLRs in Jade perch. These data will make a contribution for better understanding the antibacterial mechanism of TLRs in teleosts.