Multi-sequence Alignment Research Articles

Cloning and Characterization of Eukaryotic Translation Initiation Factor 4E (eIF4E) Gene Family in <i>Ipomoea batatas</i> L. (Lam) for Understanding Hexaploid Sweetpotato-Virus Interactions

Characterization of genes related to sweetpotato to viral disease resistance is critical for understanding plant-pathogen interactions, especially with feathery mottle virus infection. For example, genes encoding eukaryotic translation initiation factor (eIF)4E, its isoforms, eIF(iso)4E, and the cap-binding protein (CBP) in plants, have been implicated in viral infections aside from their importance in protein synthesis. Full-length cDNA encoding these putative eIF targets from susceptible/resistant and unknown hexaploid sweetpotato (Ipomoea batatas L. Lam) were amplified based on primers designed from the diploid wild-type relative Ipomoea trifida consensus sequences, and designated IbeIF4E, IbeIF(iso)4E and IbCBP. Comparative analyses following direct-sequencing of PCR-amplified cDNAs versus the cloned cDNA sequences identified multiple homeoalleles: one to four IbeIF4E, two to three IbeIF(iso)4E, and two IbCBP within all cultivars tested. Open reading frames were in the length of 696 bp IbeIF4E, 606 bp IbeIF(iso)4E, and 675 bp IbCBP. The encoded single polypeptide lengths were 232, 202, and 225 amino acids for IbeIF4E, IbeIF(iso)4E, and IbCBP, with a calculated protein molecular mass of 26 kDa, 22.8 kDa, and 25.8 kDa, while their theoretical isoelectric points were 5.1, 5.57, and 6.6, respectively. Although the homeoalleles had similar sequence lengths, single nucleotide polymorphisms and multi-allelic variations were detected within the coding sequences. The multi-sequence alignment performed revealed a 66.9% - 96.7% sequence similarity between the predicted amino acid sequences obtained from the homeoalleles and closely related species. Furthermore, phylogenetic analysis revealed ancestral relationships between the eIF4E homeoalleles and other species. The outcome herein on the eIF4E superfamily and its correlation in sequence variations suggest opportunities to decipher the role of eIF4E in hexaploid sweetpotato feathery mottle virus infection.

Genome-wide identification and expression analysis of ethylene responsive factor family transcription factors in Juglans regia.

BackgroundWalnut is an important economic tree species with prominent economic value and ecological functions. However, in recent years, walnuts have become susceptible to drought stress, resulting in a decline in comprehensive benefits. Therefore, it is necessary to identify the regulatory molecular mechanism associated with walnut response to drought. In many plants, ethylene responsive factor (ERF) gene family plays important roles in response to biotic and abiotic stress, especial drought. Therefore, the identification and characterisation of walnut ERF genes will benefit walnut with regard to the clarification of drought response mechanism as well as the management, production, and quality of plantations.Methods‘ERF’ was compared against the walnut transcriptome, and the JrERFs with a complete open reading frame (ORF) were identified by ORF Finder. The molecular weights, amino acid residues, and theoretical isoelectric point (pI) were predicted by ExPASy. The distribution of JrERFs in chromosome locations was determined based on walnut genome data from NCBI. The intron-exon structures and conserved domains were analysed using Gene Structure Display Server 2.0 and CD-Search, accordingly. Multi-sequence alignment and a phylogenetic tree were constructed by ClustalX2.1 and MEGA7, respectively. The conserved motifs were acquired using MEME. Total RNA was isolated using the cetyltrimethylammonium ammonium bromide (CTAB) method (Yang et al., 2018). Gene expression was determined by using real-time quantitative polymerase chain reaction (qRT-PCR) analysis and calculated according to the 2−ΔΔCT method (Livak & Schmittgen, 2001).ResultsA total of 44 JrERFs were identified from the walnut transcriptome, whose ORFs were 450–1,239 bp in length. The molecular weights of the JrERF proteins (consisting 149–412 amino acids) were 16.81–43.71 kDa, with pI ranging from 4.8 (JrERF11) to 9.89 (JrERF03). The JrERFs can be divided into six groups (B1–B6), and among the groups, B6 contained the most number of members. Each JrERF contained 1–6 motifs and each motif comprised 9–50 amino acids. Among the motifs, motif1, motif2, and motif3 were the most abundant. More than 40% of JrERFs were up-regulated continuously when subjected to ethephon (ETH), PEG6000, and PEG6000+ETH treatments. Of all the JrERFs, JrERF11 showed the highest expression. Therefore, we conclude that walnut ERF genes are highly conserved and involved in the regulation of drought response in the presence of ETH. JrERFs are possibly important candidate genes for molecular breeding; hence, the findings of this study provides the theoretical basis for further investigation of ERF genes in walnut and other species.

Extracting process hierarchies by multi-sequence alignment adaptations

ABSTRACT Process mining is an active research area that provides a wide range of automated process discovery, conformance checking and process enhancement solutions by extracting process information from event logs. With the emerge of new shared economy models and system architectures, monolithic perspective of process mining, i.e. a single process within a single organisation, is evolved towards cross-organisational business processes. The results can be used to form new collaboration among the organisations and share best practices, business knowledge or a common infrastructure. However, the variability across the cross-organisational process variants constitutes a challenge to deal with, especially at flexible environments. This paper demonstrates a quantitative approach to measure how similar organisations execute the same process. The approach is based on the multi-sequence alignment of dominant behaviour, which is a typical sequence of behaviour recurring at the process cases. Then process clusters are extracted in the form of process hierarchy according to the degree of process similarities. A novel feature of the proposed approach is that branching in process hierarchy reflects how organisations’ behaviours differ from each other. Additionally, common fragments at the aligned process variants within the same cluster pinpoint a functional inheritance or a shared process behaviour for the corresponding organisations. The proposed approach is applied on a real-life case and the comparison with previous alignment techniques reveals the advantages of the approach.

Cloning, expression, purification, and biochemical characterization of CpxR protein from pectobacterium carotovorum.

The cpxR gene, encoding a new cytoplasmic response regulator, which effects virulence, biofilm formation, chemotaxis, resistance to antimicrobials, and controls soft rot, was amplified by the polymerase chain reaction, cloned into the prokaryotic expression vector pET-15b, and expressed through the induction of isopropyl-β-d-thiogalactoside in Escherichia coli BL21 (DE3). Then, highly purified and stable CpxR protein was produced by nickel affinity chromatography and fast protein liquid chromatography, digested by thrombin and identified by Western blotting. Furthermore, the structure of the CpxR protein was estimated by circular dichroism spectroscopy and SWISS-MODEL. The CpxR protein was a functional part in signal transduction and bacterial resistance for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The resear ch of the protein stability indicated the CpxR protein had excellent thermal stability and was suitable for crystallization. Then the small crystals of CpxR protein were found in the crystallizing tank. The latest 34 cpxR sequences from the public database were selected and analyzed by molecular clustering and multisequence alignment. These cpxR sequences were roughly divided into four categories. Theseresultslaidan importantfoundationforthe furtherstructural studyoftheCpxR protein.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock

Adventitious root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

Can ACE2 Receptor Polymorphism Predict Species Susceptibility to SARS-CoV-2?

A novel severe acute respiratory syndrome coronavirus, SARS-CoV-2, emerged in China in December 2019 and spread worldwide, causing more than 1.3 million deaths in 11 months. Similar to the human SARS-CoV, SARS-CoV-2 shares strong sequence homologies with a sarbecovirus circulating in Rhinolophus affinis bats. Because bats are expected to be able to transmit their coronaviruses to intermediate animal hosts that in turn are a source of viruses able to cross species barriers and infect humans (so-called spillover model), the identification of an intermediate animal reservoir was the subject of intense researches. It was claimed that a reptile (Ophiophagus hannah) was the intermediate host. This hypothesis was quickly ruled out and replaced by the pangolin (Manis javanica) hypothesis. Yet, pangolin was also recently exonerated from SARS-CoV-2 transmission to humans, leaving other animal species as presumed guilty. Guided by the spillover model, several laboratories investigated in silico the species polymorphism of the angiotensin I converting enzyme 2 (ACE2) to find the best fits with the SARS-CoV-2 spike receptor-binding site. Following the same strategy, we used multi-sequence alignment, 3-D structure analysis, and electrostatic potential surface generation of ACE2 variants to predict their binding capacity to SARS-CoV-2. We report evidence that such simple in silico investigation is a powerful tool to quickly screen which species are potentially susceptible to SARS-CoV-2. However, possible receptor binding does not necessarily lead to successful replication in host. Therefore, we also discuss here the limitations of these in silico approaches in our quest on the origins of COVID-19 pandemic.

Deciphering the Subtype Differentiation History of SARS-CoV-2 Based on a New Breadth-First Searching Optimized Alignment Method Over a Global Data Set of 24,768 Sequences.

SARS-CoV-2 has caused a worldwide pandemic. Existing research on coronavirus mutations is based on small data sets, and multiple sequence alignment using a global-scale data set has yet to be conducted. Statistical analysis of integral mutations and global spread are necessary and could help improve primer design for nucleic acid diagnosis and vaccine development. Here, we optimized multiple sequence alignment using a conserved sequence search algorithm to align 24,768 sequences from the GISAID data set. A phylogenetic tree was constructed using the maximum likelihood (ML) method. Coronavirus subtypes were analyzed via t-SNE clustering. We performed haplotype network analysis and t-SNE clustering to analyze the coronavirus origin and spread. Overall, we identified 33 sense, 17 nonsense, 79 amino acid loss, and 4 amino acid insertion mutations in full-length open reading frames. Phylogenetic trees were successfully constructed and samples clustered into subtypes. The COVID-19 pandemic differed among countries and continents. Samples from the United States and western Europe were more diverse, and those from China and Asia mainly contained specific subtypes. Clades G/GH/GR are more likely to be the origin clades of SARS-CoV-2 compared with clades S/L/V. Conserved sequence searches can be used to segment long sequences, making large-scale multisequence alignment possible, facilitating more comprehensive gene mutation analysis. Mutation analysis of the SARS-CoV-2 can inform primer design for nucleic acid diagnosis to improve virus detection efficiency. In addition, research into the characteristics of viral spread and relationships among geographic regions can help formulate health policies and reduce the increase of imported cases.

Identification and Characterization of a Periplasmic α-Carbonic Anhydrase (CA) in the Gametophytes of Saccharina japonica (Phaeophyceae).

Periplasmic or external carbonic anhydrases (CAs) have been well accepted as playing a crucial role in the acquisition of dissolved inorganic carbon; however, no cytological evidence or molecular information on these enzymes has been reported in seaweeds to date. In this study, the full-length cDNA sequence coding for a putative periplasmic Sjα-CA2 was cloned from the gametophytes of Saccharina japonica, an industrial brown seaweed. It was 1,728bp in length and included a 263-bp 5'-untranslated region (UTR), a 577-bp 3'-UTR, and an 888-bp open reading frame encoding a protein precursor consisting of 295 amino acids. The mature protein, after removal of a predicted 28-residue signal peptide, was composed of 267 amino acids with a relative molecular weight of 29.27kDa. Multisequence alignment and phylogenetic analysis indicated that it was a member of the α-CA family. Enzyme activity assays showed that the recombinant Sjα-CA2 in Escherichia coli possessed CO2 hydration and esterase activities, thus identifying this gene Sjα-CA2 in function. Immunogold electron microscopic observations with the prepared anti-Sjα-CA2 polyclonal antibody illustrated that Sjα-CA2 was located in periplasmic space of the kelp gametophyte cells. Quantitative real-time PCR results revealed that the transcription of Sjα-CA2 was induced by elevated levels, but it was little changed while the kelp gametophytes were subjected to elevated CO2 concentrations. This study suggests that the periplasmic Sjα-CA2 might play a role in adapting to elevated environmental levels of by dehydration of to generate CO2 , which could be readily taken up by S. japonica gametophytes.

Isolation and characterization of a γ-carbonic anhydrase localized in the mitochondria of Saccharina japonica

Saccharina japonica is an ecologically and economically important seaweed that is dominant in the rocky shores of cold-temperate regions, forms the major component of productive beds, and affects marine environments. S. japonica exhibits a high photosynthetic efficiency in natural seawater with low dissolved CO2 concentration, thus suggesting the presence of its carbon-concentrating mechanism (CCM). However, the genes, proteins, and pathways involved in the CCM of S. japonica have not been fully identified and characterized. Carbonic anhydrase (CA) is a crucial component of CCM in macroalgae. In this study, the cloning, characterization, and subcellular localization of a specific CA were described. Multisequence alignment and phylogenetic analysis indicated that this CA belonged to the gamma (Sjγ-CA) class. This enzyme has a full-length cDAN of 1370 bp, encodes a protein with 246 amino acids (aa; ca. 25.7 kDa), and contains the mitochondrial transit peptide of 16 aa and LbH_gama_CA_like domain of 159 aa that defined the γ-CA region. The Sjγ-CA was successfully expressed in E. coli BL21 and purified as an active recombinant CA. Immunogold electron microscopy and fluorescence localization illustrated that this enzyme is localized in the mitochondria, and its transcription level is up-regulated by low CO2 concentration. These findings showed that Sjγ-CA is a possible component of the CCM in S. japonica. This work is the first to report about the mtCA of macroalgae and provides a basis for further analysis on seaweed CCM.

Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline.

Infecting millions of people, the SARS‐CoV‐2 is evolving at an unprecedented rate, demanding advanced and specified analytic pipeline to capture the mutational spectra. In order to explore mutations and deletions in the spike (S) protein — the most‐discussed protein of SARS‐CoV‐2 — we comprehensively analyzed 35,750 complete S protein‐coding sequences through a custom Python‐based pipeline. This GISAID‐collected dataset of until 24 June 2020 covered six continents and five major climate zones. We identified 27,801 (77.77% sequences) mutated strains compared to reference Wuhan‐Hu‐1 wherein 84.40% of these strains mutated by only a single amino acid (aa). An outlier strain (EPI_ISL_463893) from Bosnia and Herzegovina possessed six aa substitutions. We also identified 11 residues with high aa mutation frequency, and each contains four types of aa variations. The infamous D614G variant has spread worldwide with ever‐rising dominance and across regions with different climatic conditions alongside L5F and D936Y mutants, which have been documented throughout all regions and climate zones, respectively. We also found 988 unique aa substitutions spanned across 660 residues, which differed significantly among different continents (p = .003) and climatic zones (p = .021) as inferred with the Kruskal–Wallis test. Besides, 17 in‐frame deletions at four sites adjacent to receptor‐binding‐domain were determined that may have a possible impact on attenuation. This study provides a fast and accurate pipeline for identifying mutations and deletions from the large dataset for coding and also non‐coding sequences as evidenced by the representative analysis on existing S protein data. By using separate multi‐sequence alignment, removing ambiguous sequences and in‐frame stop codons, and utilizing pairwise alignment, this method can derive both synonymous and non‐synonymous mutations (strain_ID reference aa:mutation position:strain aa). We suggest that the pipeline will aid in the evolutionary surveillance of any SARS‐CoV‐2 encoded proteins and will prove to be crucial in tracking the ever‐increasing variation of many other divergent RNA viruses in the future. The code is available at https://github.com/SShaminur/Mutation-Analysis.

Knocking out Analysis of the CpxP gene using Crispr/Cas9 in Escherichia coli MG1655

Based on the analysis of cpxP genes among Escherichia coli strains, cpxP gene-targeting short guide RNA (sgRNA) was designed and inserted into the pGL3-MGP-RNA. The donor sequences (MG-HR) for homologous repair were designed and cloned by PCR. MG-HR and pGL3-MGP-RNA were transformed into E. coli MG1655 (pCas9). The cpxP gene expression cassette was amplified by PCR and subcloned into pBBR1MCS-2. Then the pBBR-cpxP was independently transformed into E. coli MG1655. The results of motility experiment suggest that cpxP gene had a significant effect on the movement ability of E. coli strain. The CpxP protein had a significant inhibition of bacterial activity. The lastest 81 CpxP proteins sequences were selected and analyzed by multi-sequence alignment and molecular cluster. The CpxP proteins were roughly divided into three categories. Our results suggest that the CpxP protein was involved in bacterial motility, infection and pathogenicity.

Immunocytochemical Localization of the Kinetochore Protein Nuf2p on the Gametophyte Chromosomes of a Cultivar of Saccharina (Phaeophyta)

Nuf2p, a centromere-associated protein, is an indispensable component of Ndc80 complex which is important for stable microtubule-kinetochore attachment, chromosome pairing and spindle assembly in mitosis. Based upon contig sequences coding for Nuf2p from a pyrosequencing transcriptome of Saccharina japonica, primers were designed for molecular cloning of this kinetochore protein. The open reading frame (ORF) of this gene SjNuf2 was 1,452 bp long, encoding a putative protein consisting of 483 amino acids. Multi-sequence alignment showed SjNuf2p possessed several conserved domains of Nuf2, Spc7, SPT2 and MIT. To illustrate the location of SjNuf2p using immunocytochemistry, its ORF was inserted into a prokaryotic expression vector pET-28a. Then the recombinant SjNuf2p (rSjNuf2p) was induced to be expressed in Escherichia coli. The rSjNuf2p was verified by Western blotting with the commercial anti-6His-tag antibody and then purified using affinity chromatography. The polyclonal antibody of anti-SjNuf2p was raised against the purified rSjNuf2p. Western blotting with this prepared antibody showed the presence of SjNuf2p in the gametophytes of a Saccharina cultivar. SjNuf2p and alpha/beta-tubulin were subsequently co-localized to the paraffin sections of kelp chromosomes with their corresponding antibodies, thus illustrating that SjNuf2p could reside at the kelp centromeres. This study laid a solid foundation for kelp centromere and karyotype analyses.

First report of Godronia cassandrae as a major cranberry fruit rot pathogen in Eastern Canada.

The complex etiology of cranberry fruit rot (CFR) (Oudemans et al., 1998) has made it difficult to precisely identify the fungi involved in CFR and their relative importance in North America. To remedy this situation, a multiplex PCR approach targeting the 12 most commonly reported fungi in CFR was recently developed (Conti et al., 2019). However, in surveys conducted in Eastern Canada, the molecular tool revealed the presence of an unknown fungus in more than 30% of the collected samples. Analyses were thus undertaken to identify this species. From 117 rotten fruit collected at harvest in 2017, 34 samples of the unknown fungus, all morphologically similar, were isolated but not detected using the molecular tool. Their ITS ribosomal regions were sequenced using universal primers (Vilgalys and Hester, 1990; White et al., 1990) and searched against the GenBank database using the Blastn tool (Altschul et al., 1990). The top match was obtained with Godronia cassandrae (accession number: MH855281 (Vu et al., 2019), 98-100% of identity and an E-value of 0.0), even though some isolates had minor nucleotide differences, as presented in the tree. Sequences were deposited in GenBank as accession numbers MT599989 to MT600022. Since G. cassandrae had been reported, albeit rarely, on cranberry in Michigan (Olatinwo et al., 2003), it was supposed to amplify with the molecular tool developed from the strain DAOM C216021 (AAFC, Ottawa, ON) identified in 1993 on Vaccinium angustifolium as G. cassandrae. Analysis of the sequences used to build the specific primers from this strain confirmed the DAOM strain as being Neocucurbitaria juglandicola, which was never diagnosed in our cranberry samples. To confirm this revised diagnosis, a multi-sequence alignment (MSA) was performed on the ITS regions of the isolates from rotten cranberries and sequences available for the genus Godronia in the NCBI nucleotide database (NCBI txid269064). This MSA allowed us to find discriminant regions between Godronia spp. A pair of PCR primers specific to G. cassandrae found on cranberry fruit was then designed (the forward and reverse sequences are AAT CAG TGG CGG TGC CTG TC and TAC CGC TTC ACT CGC CGT TAC, respectively), generating 196 bp amplicons, with an annealing temperature of 65°C. The diagnosis of 7,835 fruit sampled at three time points (harvest, after three and after six weeks of storage) in 2018, from four cranberry farms located in Québec (CA) and Nova Scotia (CA), detected G. cassandrae in 2350 samples (30%). To assess the pathogenicity of four specimens from 2017, Koch's postulates were completed on two healthy fruit per isolate. The fruit were wounded with a sterilized pick and individually inoculated; two fruit were used as control. Based on our observations, the fungi isolated from cranberry fruit displayed a pale lemon yellow mycelium and black pycnidia. Conidia are hyaline, cylindrical and divided by a single septum. These morphological characters are similar to the ones described in the literature for G. cassandrae (Polashock et al., 2017). Rot symptoms appear as a discoloration from the firm, red and healthy cranberry fruit to a yellowish-orange softer fruit. Molecular characterization of the re-isolated fungus confirmed the presence of G. cassandrae. We report Godronia cassandrae for the first time as a major cause of CFR in Eastern Canada. Its prevalence in cranberry fields of Québec and Nova Scotia suggests that it supplants Physalospora as the main fungus involved in CFR in Eastern Canada.

Comparative Genome Analysis of Bacillus sporothermodurans with Its Closest Phylogenetic Neighbor, Bacillus oleronius, and Bacillus cereus and Bacillus subtilis Groups.

Bacillus sporothermodurans currently possesses one of the most highly heat-resistant spores (HRS), which can withstand ultra-high temperature (UHT) processing. Determination of multiple whole genome sequences of B. sporothermodurans provided an opportunity to perform the first comparative genome analysis between strains and with B. oleronius, B. cereus, and B. subtilis groups. In this study, five whole genome sequences of B. sporothermodurans strains, including those belonging to the HRS clone (SAD and BR12) normally isolated from UHT milk, were compared with the aforementioned Bacillus species for gene clusters responsible for heat resistance. In the phylogenomic analysis, B. sporothermodurans, with its closest phylogenetic neighbor, B. oleronius, clustered with B. thermoamylovorans and B. thermotolerans. Heat shock proteins GrpE, GroES, GroEL, and DnaK presented identical sequences for all B. sporothermodurans strains, indicating that differences in functional efficiency are not involved in the thermal resistance variations. However, comparing all species evaluated, B. sporothermodurans exhibited a different gene configuration in the chromosomal region of the heat shock protein GrpE. Furthermore, only B. sporothermodurans strains presented the stage II sporulation protein P gene located in this region. Multisequence alignment and phylogenetic analysis of the ClpB protein showed differences for HRS and non-HRS strains. The study identified ClpC, ClpE, and ClpX as the three ATPases putatively involved in protein disaggregation in B. sporothermodurans. Bacillus sporothermodurans exhibits high homology with other Bacillus species in the DnaK, DnaJ, GroEL, and GroES cluster of genes involved in heat resistance. The data presented here pave the way to select and evaluate the phenotypic effects of genes putatively involved in heat resistance.

Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata

BackgroundThe recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages.ResultsIn this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1.ConclusionsThis study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

Bioinformatics analysis reveals four major hexon variants of human adenovirus type-3 (HAdV-3) as the potential strains for development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections

Human adenovirus type 3 (HAdV-3) encompasses 15–87% of all adenoviral respiratory infections. The significant morbidity and mortality, especially among the neonates and immunosuppressed patients, demand the need for a vaccine or a targeted antiviral against this type. However, due to the existence of multiple hexon variants (3Hv-1 to 3Hv-25), the selection of vaccine strains of HAdV-3 is challenging. This study was designed to evaluate HAdV-3 hexon variants for the selection of potential vaccine candidates and the use of hexon gene as a target for designing siRNA that can be used as a therapy. Based on the data of worldwide distribution, duration of circulation, co-circulation and their percentage among all the variants, 3Hv-1 to 3Hv-4 were categorized as the major hexon variants. Phylogenetic analysis and the percentage of homology in the hypervariable regions followed by multi-sequence alignment, zPicture analysis and restriction enzyme analysis were carried out. In the phylogram, the variants were arranged in different clusters. The HVR encoding regions of hexon of 3Hv-1 to 3Hv-4 showed 16 point mutations resulting in 12 amino acids substitutions. The homology in HVRs was 81.81–100%. Therefore, the major hexon variants are substantially different from each other which justifies their inclusion as the potential vaccine candidates. Interestingly, despite the significant differences in the DNA sequence, there were many conserved areas in the HVRs, and we have designed functional siRNAs form those locations. We have also designed immunogenic vaccine peptide epitopes from the hexon protein using bioinformatics prediction tool. We hope that our developed siRNAs and immunogenic vaccine peptide epitopes could be used in the future development of siRNA-based therapy and designing a vaccine against HAdV-3.

Genome wide analysis and functional identification of MdKCS genes in apple

Apple fruit is covered by cuticle wax, which plays important roles protecting fruits from adverse environmental conditions. β-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme in plant wax synthesis. In this study, we identified 28 KCS gene family members from apple (Malus × domestica Borkh.) by homology analysis. Multi-sequence alignment and phylogenetic analyses revealed that the 28 MdKCS genes were divided into four subgroups, including KCS1-like, FAE1-like, FDH-like, and CER6. A chromosomal localization analysis revealed that 27 apple KCS genes were located on 11 chromosomes, while MdKCS28 was localized to the unassembled genomic scaffold. Most of the MdKCS proteins were hydrophilic proteins and they had similar secondary and tertiary structures. The prediction of cis-acting elements of the MdKCS gene promoters suggested that the MdKCS genes may be widely involved in hormone signaling and the stress response. Furthermore, the quantitative real-time polymerase chain reaction results showed that eight MdKCS genes were highly expressed in the apple pericarp, and were significantly induced by drought, abscisic acid (ABA), and NaCl treatments. We transformed the MdKCS21 gene into apple calli, and found the MdKCS21 overexpressing transgenic apple calli exhibited higher tolerance to ABA treatment. Finally, the MdKCS proteins were localized to the endoplasmic reticulum and vacuolar membrane by confocal laser microscopy. This study established a foundation to further analyze the function of KCS genes and provided candidate genes for molecular improvement of wax content in apple.

Cloning and Expression Analysis of Cdc25 Gene of <i>Sipunculus nudus</i> in Oocytes

Cell division cycle 25 (Cdc25) is an important dual specificity phosphatase, which plays an important role in regulating the process of oocyte meiosis and embryo development. In this study, the full-length cDNA of Sn-Cdc25 was cloned from S. nudus using RACE technology. The results show that Sn-Cdc25 is 4 130 bp in length, including 3′ UTR 1 849 bp and 5′ UTR 427 bp. The Open reading frame (ORF) is 1 854 bp and encodes 617 amino acids. Sequence analysis shows that the molecular weight of Sn-Cdc25 protein is 69.58 kD, with two typical Cdc25 protein domains: M-phase inducer phosphatase domain and Rhodanese-like domain, and the active site sequence HCX 5 R that can catalyze the dephosphorylation process. Multi-sequence alignment finds that the C-terminal homology is higher than the N-terminal. The tertiary structure prediction shows that the spatial conformation of Cdc25 homologous protein and its active site are highly conservative. A total of 5 Motifs are found in Motif analysis, of which Motif 1 and Motif 2 are Paxillin LD motif and MYND domain binding motif, respectively. Phylogenetic tree analysis shows that Cdc25 is clustered into two branches: invertebrates and vertebrates. RT-PCR results show that the expression of Sn-Cdc25 , with two peaks, is significantly different in different developmental stages of oocytes. The increase in the expression of Sn-Cdc25 from primary vitellogenic stage to the late of active vitellogenic stage (O1-O3) may be related to the process of Sn-Cdc25 promoting DNA replication. When the oocytes entering metanephridium from coelomic fluid, the rapid rise of Sn-Cdc25 expression may be beneficial to the activation of maturation promoting factor (MPF). The above results have accumulated basic data for further understanding of the developmental mechanism of Sipuncula oocytes and for optimization of artificial breeding techniques.

The human nephrin Y1139RSL motif is essential for podocyte foot process organization and slit diaphragm formation during glomerular development

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.

CUDAMPF++: A Proactive Resource Exhaustion Scheme for Accelerating Homologous Sequence Search on CUDA-Enabled GPU

Genomic sequence alignment is an important research topic in bioinformatics and continues to attract significant efforts. As genomic data grow exponentially, however, most of alignment methods face challenges due to their huge computational costs. HMMER, a suite of bioinformatics tools, is widely used for the analysis of homologous protein and nucleotide sequences with high sensitivity, based on profile hidden Markov models (HMMs). Its latest version, HMMER3, introdues a heuristic pipeline to accelerate the alignment process, which is carried out on central processing units (CPUs) with the support of streaming SIMD extensions (SSE) instructions. Few acceleration results have since been reported based on HMMER3. In this paper, we propose a five-tiered parallel framework, CUDAMPF++, to accelerate the most computationally intensive stages of HMMER3's pipeline, multiple/single segment Viterbi (MSV/SSV), on a single graphics processing unit (GPU). As an architecture-aware design, the proposed framework aims to fully utilize hardware resources via exploiting finer-grained parallelism (multi-sequence alignment) compared with its predecessor (CUDAMPF). In addition, we propose a novel method that proactively sacrifices L1 Cache Hit Ratio (CHR) to get improved performance and scalability in return. A comprehensive evaluation shows that the proposed framework outperfroms all existig work and exhibits good consistency in performance regardless of the variation of query models or protein sequence datasets. For MSV (SSV) kernels, the peak performance of the CUDAMPF++ is 283.9 (471.7) GCUPS on a single K40 GPU, and impressive speedups ranging from 1.x (1.7x) to 168.3x (160.7x) are achieved over the CPU-based implementation (16 cores, 32 threads).