Aa Residues Research Articles

Compounds Identified from Marine Mangrove Plant (Avicennia alba) as Potential Antiviral Drug Candidates Against WDSV, an In-Silico Approach.

Walleye dermal sarcoma virus (WDSV) is a type of retrovirus, which affects most of the adult walleye fishes during the spawning time. The virus causes multiple epithelial tumors on the fish’s skin and fins that are liable for more than 50% of the mortality rate of fish around the world. Till now, no effective antiviral drug or vaccine candidates have been developed that can block the progression of the disease caused by the pathogen. It was found that the 582-amino-acid (aa) residues long internal structural gag polyprotein of the virus plays an important role in virus budding and virion maturation outside of the cell. Inhibition of the protein can block the budding and virion maturation process and can be developed as an antiviral drug candidate against the virus. Therefore, the study aimed to identify potential natural antiviral drug candidates from the tropical mangrove marine plant Avicennia alba, which will be able to block the budding and virion maturation process by inhibiting the activity of the gag protein of the virus. Initially, a homology modeling approach was applied to identify the 3D structure, followed by refinement and validation of the protein. The refined protein structures were then utilized for molecular docking simulation. Eleven phytochemical compounds have been isolated from the marine plant and docked against the virus gag polyprotein. Three compounds, namely Friedlein (CID244297), Phytosterols (CID12303662), and 1-Triacontanol (CID68972) have been selected based on their docking score −8.5 kcal/mol, −8.0 kcal/mol and −7.9 kcal/mol, respectively, and were evaluated through ADME (Absorption, Distribution, Metabolism and Excretion), and toxicity properties. Finally, molecular dynamics (MD) simulation was applied to confirm the binding stability of the protein-ligands complex structure. The ADME and toxicity analysis reveal the efficacy and non-toxic properties of the compounds, where MD simulation confirmed the binding stability of the selected three compounds with the targeted protein. This computational study revealed the virtuous value of the selected three compounds against the targeted gag polyprotein and will be effective and promising antiviral candidates against the pathogen in a significant and worthwhile manner. Although in vitro and in vivo study is required for further evaluation of the compounds against the targeted protein.

Genetic Diversity of Recent Infectious Bursal Disease Viruses Isolated From Vaccinated Poultry Flocks in Malaysia.

Vaccination is an essential component in controlling infectious bursal disease (IBD), however, there is a lack of information on the genetic characteristics of a recent infectious bursal disease virus (IBDV) that was isolated from IBD vaccinated commercial flocks in Malaysia. The present study investigated 11 IBDV isolates that were isolated from commercial poultry farms. The isolates were detected using reverse transcription-polymerase chain reaction (RT-PCR) targeting the hypervariable region (HVR) of VP2. Based on the HVR sequences, five isolates (IBS536/2017, IBS624/2017, UPM766/2018, UPM1056/2018, and UPM1432/2019) were selected for whole-genome sequencing using the MiSeq platform. The nucleotide and amino acid (aa) sequences were compared with the previously characterized IBDV strains. Deduced aa sequences of VP2HVR revealed seven isolates with 94–99% aa identity to very virulent strains (genogroup 3), two isolates with 97–100% aa identity to variant strains (genogroup 2), and two strains with 100% identity to the vaccine strain (genogroup 1) of IBDV. The phylogenetic analysis also showed that the isolates formed clusters with the respective genogroups. The characteristic motifs 222T, 249K, 286I, and 318D are typical of the variant strain and were observed for UPM1219/2019 and UPM1432/2019. In comparison, very virulent residues such as 222A, 249Q, 286T, and 318G were found for the vvIBDV, except for the UPM1056/2018 strain with a A222T substitution. In addition, the isolate has aa substitutions such as D213N, G254D, S315T, S317R, and A321E that are not commonly found in previously reported vvIBDV strains. Unlike the other vvIBDVs characterized in this study, UPM766/2018 lacks the MLSL aa residues in VP5. The aa tripeptides 145/146/147 (TDN) of VP1 were conserved for the vvIBDV, while a different motif, NED, was observed for the Malaysian variant strain. The phylogenetic tree showed that the IBDV variant clustered with the American and Chinese variant viruses and are highly comparable to the novel Chinese variants, with 99.9% identity. Based on the sequences and phylogenetic analyses, this is the first identification of an IBDV variant being reported in Malaysia. Further research is required to determine the pathogenicity of the IBDV variant and the protective efficacy of the current IBD vaccines being used against the virus.

Two strains of a novel begomovirus encoding Rep proteins with identical β1 strands but different β5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the β-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Kefir peptides exhibit antidepressant-like activity in mice through the BDNF/TrkB pathway

Depression is a prevalent, stress-related mental disorder that can lead to serious psychiatric diseases with morbidity and high mortality. Although some functional fermented dairy drinks have promising anxiolytic and antidepressant effects, the mechanism is still not clear. To determine the antidepressant-like effect and the potential molecule mechanism of kefir peptides (KP), various behavioral tests, including the elevated plus maze test, open field test, forced swimming test, and tail suspension test, were used. Administration of 150 mg/kg KP in mice reduced the duration of immobility in the forced swimming test and tail suspension test, elevated the time spent in the open arm and center zone in the elevated plus maze test, and increased the total distance traveled, average speed, and time spent in the center zone in the open field test compared with the mock group. These results indicated that KP dramatically ameliorated the depression-like behaviors. Kefir peptides were further isolated and identified using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, from which 3 peptides were identified and designated KFP-1, KFP-3, and KFP-5. Among these peptides, administration of KFP-3 (15 AA residues) remarkably decreased immobility time in the forced swimming test and increased mobility time in the tail suspension test. Therefore, KFP-3 may be the major active peptide with antidepressant activity in KP. Overexpression of brain-derived neurotrophic factor, phosphorylated tropomyosin receptor kinase B, and phosphorylated ERK1/2 protein levels could be detected in the hippocampus under KP administration. Therefore, we suggest that KP improves depressive-like behaviors by activating the brain-derived neurotrophic factor-phosphorylated tropomyosin receptor kinase B signaling pathway. Kefir peptides may serve as a new type of antidepressant dairy product and may provide potent antidepressant effects for clinical use.

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

Novel bacteriocins produced by Lactobacillus fermentum strains with bacteriostatic effects in milk against selected indicator microorganisms

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.

The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold.

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVRA1, AVRA7, AVRA9, AVRA13, and allelic AVRA10/AVRA22, which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVRA6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVRA1, AVRA7, AVRA10/AVRA22, and AVRA13, suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVRA1 and AVRA6 as well as allelic AVRA10 and AVRA22 is largely determined by the receptors’ C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVRA10 and five specific AVRA22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVRA effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.

Association of Mutations in the NS5A-PKRBD Region and IFNL4 Genotypes with Hepatitis C Interferon Responsiveness and its Functional and Structural Analysis

Background: The cellular antiviral responses induced by interferons require some cellular protein kinase for its activation. Evidence indicated that a number of Hepatitis C Virus (HCV) proteins can repress double-stranded (ds) RNA-dependent Protein Kinase (PKR) function and help HCV to escape. However, the reports are controversial, some researchers have suggested that a region in Nonstructural 5A (NS5A) gene called Protein Kinase R-Binding Domain (PKR-BD) is associated with HCV sensitivity to the antiviral effects of Interferon (IFN). In addition, the other factor that might be associated with response to PEGylated-IFNα (Peg-IFNα) and Ribavirin (RBV) combination therapy, is IFNL4 genotypes. Objective: The aim of this study was to investigate the association between amino acid (aa) substitutions in the NS5A region and the IFNL4 genotypes in two Single Nucleotide Polymorphism (SNP) (rs8099917. rs12979860) in patients with HCV genotypes 1a and 3a. We also examined their response to combination therapy and the effect of these mutations on the function and structure of PKR-BD. Methods: Eighty-six patients with hepatitis C were recruited and follow-up for 6 months. Several tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), viral load, IFNL4 genotyping, and PKR-BD sequencing were performed. Using several well-known and trustworthy bioinformatics tools, sequences were analyzed to define physio-chemical properties, structural features, immune epitopes and protein-protein interaction. Results: Of the 86 patients, 65.1% had high viral load at baseline, 64% had CT genotype for rs12979860 and 57% had GT genotype for rs8099917. Several aa residues changes were found in the PKR-BD region. We could not find any link between mutations in the PKR-BD region and different genotypes of IFNL4 in response to antiviral therapy. Regardless of pI, PKR-BD 1a and 3a showed similar physio-chemical properties, and 2 phosphorylation sites and one glycosylation site were estimated for both PKR-BD 1a and 3a. Trustworthy software were employed in order to predict B-cell epitopes, 3 regions (6-17, 26-32, 34-41) were found for both proteins, indicating a huge potential of PKR-BD protein to induce humoral immune system. Docking analysis determined non-responder sequences in both 1a and 3a genotypes to have higher energy value and are more compatible with PKR. Conclusion: To sum up, our results could not determine any significant relationship between mutations of PKR-BD and genotypes of IFNL4 with other factors; ALT, AST, viral load. However, docking results showed strengthened interaction between PKR-BD and PKR in non-responders that could have a momentous impact on the illness severity.

First report of fig mosaic virus on fig in Russia.

Fig mosaic virus (FMV) (genus Emaravirus in the family Fimoviridae) is considered the etiological agent of fig mosaic disease (FMD) that is recorded in most of the fig growing areas with an average global infection rate of 33%. The multipartite FMV genome is comprised of six negative monocistronic ssRNAs, each of which is separately encapsidated (Preising et al. 2020). Although FMD-like symptoms, which include mosaic, chlorotic ringspots, and oak leaf patterns, were observed in approximately a third of 400 fig accessions in the Nikita Botanical Gardens, Yalta, Russia (Mitrofanova et al. 2016), FMV has not been identified as the causal agent of the disease. In June of 2020, total RNA was isolated from symptomatic leaves of 59 thirty two-year-old trees representing 31 local and 27 introduced Ficus carica L. cultivars and a single F. pseudocarica Miq. tree using RNeasy Plant Mini kit (Qiagen, USA). FMV was tested by RT-PCR using primer sets E5 (Elbeaino et al. 2009) and EMARAVGP (Walia et al. 2009), which amplify a 302-bp fragment of RNA1 and a 468-bp fragment of RNA2, respectively. PCR products of the expected sizes were generated in all samples, indicating a high FMV incidence in the plantings. The genome sequences of FMV isolates from F. carica cvs. Bleuet, Kraps di Hersh, Smena, Temri, and F. pseudocarica (Fig. S1) were determined by high-throughput sequencing on MiSec Illumina platform. Double-stranded RNA was isolated from FMV-positive leaves using Viral Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, USA). In average, 695,000 quality-filtered 150 bp pair-ended reads per a library were produced and used in a de novo assembly using metaSpades program version 3.14 (Nurk et al. 2017). In each of five samples, BLASTn analysis found six FMV-related contigs. The contigs spanned 99 to 100% of corresponding genomic segments of the most closely related isolates. In addition to FMV, fig cryptic virus-related contigs were also detected in some samples. The FMV contigs covering RNA1 to RNA6 had the highest identity to corresponding genomic segments of isolates AM941711 (96.5 to 96.6%), FM864225 (94.4 to 94.6%), FM991954 (97.9 to 98.2%), AB697863 (96.4 to 96.6%), AB697879 (93.3 to 93.4%), and AB697895 (95.4 to 97.0%), respectively. Five Russian isolates shared 99.2 to 100% nucleotide sequence identity, depending on the genomic segment. Their sequences were deposited in GenBank under accession numbers MW201216 to MW201230 and MW208662 to MW208676. Phylogenetic analysis of six ORFs showed that ORF1 to ORF3 and ORF6 of the Russian isolates clustered with FMV isolates from Italy while ORF4 grouped with the isolate JTT-Pa (AB697863) from Japan (Fig. S2). ORF5 of the Russian isolates formed a separate cluster with the isolates SB1 and SB2 from Serbia and JTT-Vi from Japan (AB697879 to AB697884). Incongruency of phylogenetic relationship among the genomic segments suggests reassortment among ancestors of the Russian FMV isolates. In addition, similar to the SB1, SB2 and JTT-Vi, ORF5 of the Russian isolates encodes a protein of 486 amino acid (aa) residues in contrast to the corresponding protein of Italian isolates consisting of 502 aa. To the best of our knowledge, this is the first report of FMV in Russia. This finding not only expands the information on the geographical distribution of FMV, but also extends knowledge on F. pseudocarica as a natural host of the virus.

Molecular analysis of functional domain and protein motif of endoglucanase gene in marine bacteria isolated from Eucheuma sp. and Sargassum sp.

Marine bacteria which are symbionts with Eucheuma sp. and Sargassum sp. has the ability to convert seloluse into glucose. These processes are important during bioethanol production. Identification using PCR and 16S rRNA primers showed that the symbiotic bacteria in Eucheuma sp. was B. subtilis (97% identical to accession number NR. 027552.1) and symbiotic bacteria in Sargassum sp. was B. thuringiensis (97% identical to access number NR. 043403.1). Cellulotic indexes were identified for B. subtilis (2.477 mm) and B. thuringiensis (6.102 mm). Amplification of the endoglucanase gene were conducted with Bac-EuF and Bac-EuR primers in B. subtilis with size at 1416 bp (95% identical to accession number WP_017696508.1), whereas in B. thuriengeinsis the size was 1251 bp (92% identical to the accession number EEM47662). In silico analysis of the endoglucanase gene, showed that the catalytic and cellulose binding domains of B. subtilis were GH5 (aa residue 1-70) and CBM3 (aa residue 131-212), while in B. thuringiensis only GH8 catalytic domains were identified (aa residue 30 -370). The protein motif of the endoglucanase gene B. subtilis and B. thuringiensis had a high similarity characterized by the asn_glycosylation, camp_phospho_site, ck2_phospho_site, myristyl and pkc_phospho_site motif.

The evolution and functional characterization of CXC chemokines and receptors in lamprey

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense. They interact with chemokine receptors and are essential for the coordination of cell migration in diverse physiological processes. The CXC subfamily is one of the largest groups in the chemokine family and consists of multiple members. In this study, we identified homologues of three chemokine ligands (CXCL8, CXCL_F5 and CXCL12) and two CXC receptor like molecules (CXCR_L1 and CXCR_L2) in lamprey. Sequence analysis revealed that they share the same genomic organization with their counterparts in jawed vertebrates but synteny was not conserved. Lamprey CXCL8 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues. In addition, CXCL_F5 is evolutionarily related to the fish specific CXC chemokine groups previously identified and contains multiple cationic aa residues in the extended C- terminal region. The two CXCRs possess seven transmembrane domains and conserved structural elements for receptor activation and signaling, including the DRYXXI(V)Y motif in TM2, the disulphide bond connecting ECL2 and TM3, the WXP motif in TM6 and NPXXY motif in TM7. The identified CXC chemokines and receptors were constitutively expressed in tissues including the liver, kidney, intestine, heart, gills, supraneural body and primary leukocytes, but exhibited distinct expression patterns. Relatively high expression was detected in the gills for CXCL8, CXCL_F5 and CXCR_L1 and in the supraneural body for CXCL12 and CXCR_L2. All the genes except CXCL12 were upregulated by stimulation with LPS, pokeweed and bacterial infection, and the CXCL8 and CXCL_F5 was induced by poly (I:C). Functional analysis showed that the CXCL8 and CXCL_F5 specifically interacted with CXCR_L1 and CXCR_L2, respectively. Our results demonstrate that the CXC chemokine system had diversified in jawless fish.

A plant-based transient expression system for the rapid production of highly immunogenic Hepatitis E virus-like particles.

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. The aim of the study is the development of plant expression system for the production of virus-like particles formed by HEV capsid and the characterization of their immunogenicity. Open reading frame (ORF) 2 encodes the viral capsid protein and possesses candidate for vaccine production. In this study, we used truncated genotype 3 HEV ORF 2 consisting of aa residues 110 to 610. The recombinant protein was expressed in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff up to 10% of the soluble protein fraction. The yield of HEV 110-610 after purification was 150-200µg per 1g of green leaf biomass. The recombinant protein formed nanosized virus-like particles. The immunization of mice with plant-produced HEV 110-610 protein induced high levels of HEV-specific serum antibodies. HEV ORF 2 (110-610 aa) can be used as candidate for the development of a plant-produced vaccine against Hepatitis E.

An EMG-informed modelling approach to estimate muscle forces and activations in Parkinson’s disease subjects

Ucn1 was first isolated from the rat midbrain in 1995, and has a higher affinity for the type-2 CRH receptor (CRHR2) than does CRH itself. Subsequently, Ucn2 and Ucn3 were identified. Ucns have been reported in several tetrapods, teleosts, lobe-finned fish, basal ray-finned fish, cartilaginous fish, and cyclostome. Mammalian Ucn1 consists of 40 aa residues with an amidated C-terminus. In the brain, Ucn mRNA is found not only in the hypothalamus but also in the cerebral cortex and cerebellum. The gastrointestinal tract and its associated macrophages constitute a major source of Ucn in the periphery. Ucn mRNA is also abundantly detected in the hearts of humans and rodents. Ucns act through two GPCR subtypes: CRHR1 and CRHR2. CRH and Ucn1 bind to both CRHR1 and CRHR2, whereas Ucn-II and Ucn-III bind exclusively to CRHR2. Ucn1 stimulates ACTH secretion both in vivo and in vitro. Ucn1 is more potent than CRH in suppressing food intake.

Analysis of canine parvoviruses circulating in Australia reveals predominance of variant 2b and identifies feline parvovirus-like mutations in the capsid proteins.

Canine parvovirus (CPV) is a major enteric pathogen of dogs worldwide that emerged in the late 1970s from a feline parvovirus (FPV)-like ancestral virus. Shortly after its emergence, variant CPVs acquired amino acid (aa) mutations in key capsid residues, associated with biological and/or antigenic changes. This study aimed to identify and analyse CPV variants and their capsid mutations amongst Australian dogs, to gain insights into the evolution of CPV in Australia and to investigate relationships between the disease and vaccination status of dogs from which viruses were detected. CPV VP2 sequences were amplified from 79 faecal samples collected from dogs with parvoviral enteritis at 20 veterinary practices in five Australian states. The median age at diagnosis was 4months (range 1-96months). Only 3.7% of dogs with vaccination histories had completed recommended vaccination schedules, while 49% were incompletely vaccinated and 47.2% were unvaccinated. For the first time, CPV-2b has emerged as the dominant antigenic CPV variant circulating in dogs with parvoviral enteritis in Australia, comprising 54.4% of viruses, while CPV-2a and CPV-2 comprised 43.1% and 2.5%, respectively. The antigenic variant CPV-2c was not identified. Analysis of translated VP2 sequences revealed a vast repertoire of amino acid (aa) mutations. Several Australian CPV strains displayed signatures in the VP2 protein typical of Asian CPVs, suggesting possible introduction of CPV strains from Asia, and/or CPV circulation between Asia and Australia. Canine parvoviruses were identified containing aa residues typical of FPV at key capsid (VP2) positions, representing reverse mutations or residual mutations retained from CPV-2 during adaptation from an FPV-like ancestor, suggesting that evolutionary intermediates between CPV-2 and FPV are circulating in the field. Similarly, intermediates between CPV-2a-like viruses and CPV-2 were also identified. These findings help inform a better understanding of the evolution of CPV in dogs.

Symmetric benzidine derivatives as anti-HCV agents: Insight into the nature, stereochemistry of the capping amino acid and the size of the terminal capping carbamates

Novel symmetric molecules, bearing a benzidine prolinamide core, two terminal carbamate caps of variable sizes and nature, including natural and unnatural amino acids were developed. Several terminal N-carbamate substituents of the core structure, ranging from linear methyl, ethyl and butyl groups to branching isobutyl group; and an aromatic substituent were also synthesized.Series 1 has hydrophobic AA residues, namely S and R phenylglycine and a terminal carbamate capping group, whereas Series 2 bears sulphur containing amino acids, specifically S and R methionine and the natural R methylcysteine. The novel compounds were tested for their inhibitory activity (EC50) and their cytotoxicity (CC50), using an HCV 1b (Con1) reporter replicon cell line. Compound 4 with the unnatural capping residue, bearing d-Phenylglycine amino acid residue and N-isobutyloxycarbonyl capping group, was the most active within the two series, with EC50 = 0.0067 nM. Moreover, it showed high SI50 > 14788524 and was not cytotoxic at the highest tested concentration (100 μΜ), indicating its safety profile. Compound 4 also inhibited HCV genotypes 2a, 3a and 4a. Compared to the clinically approved NS5A inhibitor Daclatasvir, compound 4 shows higher activity against genotypes 1b and 3a, as well as improved safety profile.

Phosphorylation of tegument protein pp28 contributes to trafficking to the assembly compartment in human cytomegalovirus infection.

Human cytomegalovirus (HCMV) UL99 encodes a late tegument protein pp28 that is essential for envelopment and production of infectious virus. This protein is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells but it localizes to the cytoplasmic assembly compartment (AC) in HCMV-infected cells. Trafficking of pp28 to the AC is required for the assembly of infectious virus. The N-terminal domain (aa 1-61) of pp28 is sufficient for trafficking and function of the wild type protein during viral infection. However, residues required for authentic pp28 trafficking with the exception of the acidic cluster in the N-terminal domain of pp28 remain undefined. Monitoring protein migration on SDS-PAGE, we found that pp28 is phosphorylated in the virus-infected cells and dephosphorylated in the viral particles. By generating substitution mutants of pp28, we showed that three serine residues (aa 41-43) and a tyrosine residue (aa 34) account for its phosphorylation. The mutant forms of pp28 were localized to the plasma membrane as well as the ERGIC in transfected cells. Likewise, these mutant proteins were localized to the plasma membrane as well as the AC in virus-infected cells. These results suggested that phosphorylation of pp28 contributes to its intracellular trafficking and efficient viral assembly and incorporation.

Alteration of the fatty acid composition of Brassica napus L. via overexpression of phospholipid: Diacylglycerol acyltransferase 1 from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. plays an important role in traditional Chinese medicine and is one of major woody oil tree in China. Phospholipid: diacylglycerol acyltransferase 1 (PDAT1), as an important catalytic enzyme for the formation of triacylglycerol (TAG), is mainly responsible for the transfer of an acyl group from the sn-2 position of phospholipids to the sn-3 position of sn-1, 2-diacylglycerol (DAG) to produce TAG and sn-1 lysophospholipids. The importance of PDAT1 in triacylglycerol biosynthesis has been illustrated in previous research, and at least 67 PDAT1 sequences have been identified from 31 organisms. However, little is known about the gene encoding PDAT1 in S. sebiferum (SsPDAT1), which is involved in seed oil biosynthesis. To explore the functional characteristics of SsPDAT1, we cloned and analyzed the full-length cDNA in the coding region of SsPDAT1, which consists of 2040 bp and encodes a putative protein of 680 amino acid (aa) residues. Thin-layer chromatography (TLC) analysis showed that recombinant SsPDAT1 could restore TAG accumulation in TAG-deficient mutant yeast (Saccharomyces cerevisiae) H1246, which revealed the enzyme activity of SsPDAT1. Moreover, transgenic Brassica napus L. W10 plants overexpressing SsPDAT1 showed significant increases of 19.6–28.9 % in linoleic acid levels but decreases of 27.3–37.1 % in linolenic acid. Furthermore, the total oil content increased by 8.1 %–10.8 % in SsPDAT1 transgenic seeds. These results confirmed the role of SsPDAT1 in stabilizing oil biosynthesis and suggested that SsPDAT1 could be exploitable to specifically regulate the oil composition of plants. These experimental results provide a new concept that may enable the industrial development of plants with high-linoleic-acid oil through overexpression of SsPDAT1 in S. sebiferum L.

High detection rates and genetic diversity of picobirnaviruses (PBVs) in pigs on St. Kitts Island: Identification of a porcine PBV strain closely related to simian and human PBVs

We report here high rates (75.38%, 49/65) of detection of genogroup I (GI) PBVs in diarrheic pigs on the Caribbean island of St. Kitts. High quality gene segment-2 sequences encoding a significant region (~350 amino acid (aa) residues) of the putative RNA-dependent RNA polymerase (RdRp) were obtained for 23 PBV strains. The porcine PBV strains from St. Kitts exhibited high genetic diversity among themselves (deduced aa identities of 56–100%) and with other PBVs (maximum deduced aa identities of 64–97%), and retained the three domains that are conserved in putative RdRps of PBVs. The nearly complete gene segment-2 sequence (full-length minus partial 3′- untranslated region) of a porcine PBV strain (strain PO36 from St. Kitts) that is closely related (deduced aa identities of 96–97%) to simian and human GI PBVs was determined using a combination of the non-specific primer-based amplification method and conventional RT-PCR. The complete putative RdRp sequence of strain PO36 preserved the various features that are maintained in PBVs from various species. For the first time, several co-circulating PBV strains from pigs were characterized for a significant region (~350 aa) of the putative RdRp, providing important insights into the genetic diversity of PBVs in a porcine population. Taken together, these observations corroborated growing evidence that PBVs can be highly prevalent and show limited correlation globally with host species or geography. This is the first report on detection of PBVs in pigs from the Caribbean region.

A novel Kunitz-type neurotoxin peptide identified from skin secretions of the frog Amolops loloensis

A novel Kunitz-type neurotoxin peptide that inhibited voltage-gated sodium channel was purified and characterized from the skin secretions of rufous-spotted torrent frog, Amolops loloensis. It has a 240-bp cDNA encoding an 79-amino acid residue (aa) precursor protein containing 6 half-cysteines. The precursor was proven to release a 57-aa mature peptide with amino acid sequence, DRNPICNLPPKEGFCLWMMRRSFFNPSKGRCDTFGYRGCGGNKNNFETPRACKEACG. The mature was named amotoxin. Amotoxin shares sequence homology with other Kunitz-type toxins and also has three cysteine bridges. Amotoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.087 μM. To the best of our knowledge, this is the first gene-encoded neurotoxin found in Amolops loloensis. Recombinant amotoxin showed similar functional properties as the native amotoxin. The functional properties of amotoxin may provide insights into the ecological adaptation of amphibians and deepen our understanding about the biological function spectrum of amphibian skin peptides.