The discus fish (Symphysodon aequifasciata) is a cichlid demonstrating advanced mode of parental care towards fry. Both male and female fish utilized epidermal mucus secreted from specialized epidermal cells to feed developing fry. We utilized proteomics to compare protein profile from parental and nonparental fish. Gel analysis revealed a total of 35 spots that were up-regulated in parental mucus. In tandem, another 18 spots were uniquely expressed in parental mucus. MS analysis of these spots identified proteins such as fructose biphosphate aldolase, nucleoside diphosphate kinase, and heat shock proteins, which are essential to support energy provision, cell repair and proliferation, stress mediation, and defense mechanism in parental fish during parental-care period. Concurrently, the detection of several antioxidant-related proteins such as thioredoxin peroxidase and hemopexin suggests a need to overcome oxidative stress during hypermucosal production in parental-care behavior. A C-type lectin was also found to be uniquely expressed in parental mucus and could have important role in providing antimicrobial defense to both parental fish and fry. In summary, our study shows that discus mucus proteome undergoes changes in protein expression during parental-care period.
Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.
There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.
There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.
Owing to their ability to maintain a thermodynamically stable fold at extremely high temperatures, thermophilic proteins (TTPs) play a critical role in basic research and a variety of applications in the food industry. As a result, the development of computation models for rapidly and accurately identifying novel TTPs from a large number of uncharacterized protein sequences is desirable. In spite of existing computational models that have already been developed for characterizing thermophilic proteins, their performance and interpretability remain unsatisfactory. We present a novel sequence-based thermophilic protein predictor, termed SCMTPP, for improving model predictability and interpretability. First, an up-to-date and high-quality dataset consisting of 1853 TPPs and 3233 non-TPPs was compiled from published literature. Second, the SCMTPP predictor was created by combining the scoring card method (SCM) with estimated propensity scores of g-gap dipeptides. Benchmarking experiments revealed that SCMTPP had a cross-validation accuracy of 0.883, which was comparable to that of a support vector machine-based predictor (0.906-0.910) and 2-17% higher than that of commonly used machine learning models. Furthermore, SCMTPP outperformed the state-of-the-art approach (ThermoPred) on the independent test dataset, with accuracy and MCC of 0.865 and 0.731, respectively. Finally, the SCMTPP-derived propensity scores were used to elucidate the critical physicochemical properties for protein thermostability enhancement. In terms of interpretability and generalizability, comparative results showed that SCMTPP was effective for identifying and characterizing TPPs. We had implemented the proposed predictor as a user-friendly online web server at http://pmlabstack.pythonanywhere.com/SCMTPP in order to allow easy access to the model. SCMTPP is expected to be a powerful tool for facilitating community-wide efforts to identify TPPs on a large scale and guiding experimental characterization of TPPs.
Portions of the hemagglutinin neuraminidase (HN) gene of Newcastle disease virus (NDV) isolates from two recent outbreaks were sequenced to investigate epidemiology of this disease in Taiwan. These NDV isolates were all viscerotropic velogenic according to the clinical lesions produced in chickens. Sequence data were obtained from 14 NDV isolates (12 from 1995 and 2 from 1984). All isolates differed in their nucleotide sequences (from 0.3 to 15.3%), and represented potentially different strains of NDV. Phylogenetic analysis revealed that these isolates are closely related to viruses isolated from Japan and Malaysia. Some viruses isolated in 1995 appeared to evolve from viruses isolated in 1984. The results suggest that the 1995 outbreak of Newcastle disease (ND) in Taiwan may have been caused by multiple strains of velogenic NDV that have cocirculated in Taiwan for some time. Moreover, NDV isolates from racing pigeons were very similar to isolates from chickens in the same period, suggesting that both domestic and free-living birds were involved in the spread of ND in Taiwan.
1. Bradykinin and related kinins may act on four types of receptors designated as B1, B2, B3 and B4. It seems that the B2 receptors are most commonly found in various vascular and non-vascular smooth muscles, whereas B1 receptors are formed in vitro during trauma, and injury, and are found in bone tissues. 2. These BK receptors are involved in the regulations of various physiological and pathological processes. 3. The mode of kinin actions are based upon the interactions between the kinin and their specific receptors, which can lead to activation of several second-messenger systems. 4. Recently, numerous BK receptors antagonists have been synthesized with prime aim to treat diseases caused by excessive kinin production. 5. These diseases are RA, inflammatory diseases of the bowel, asthma, rhinitis and sore throat, allergic reactions, pain, inflammatory skin disorders, endotoxin and anaphylactic shock and coronary heart diseases. 6. On the other hand, BK receptor antagonists could be contraindicated in hypertension, since these drugs may antagonize the antihypertensive therapy and/or may trigger the hypertensive crisis. 7. It is worth suggesting that the BK receptor agonists might be useful antihypertensive drugs.
Phytol (PHY), a chlorophyll-derived diterpenoid, exhibits numerous pharmacological properties, including antioxidant, antimicrobial, and anticancer activities. This study evaluates the anti-diarrheal effect of phytol (PHY) along with its possible mechanism of action through in-vivo and in-silico models. The effect of PHY was investigated on castor oil-induced diarrhea in Swiss mice by using prazosin, propranolol, loperamide, and nifedipine as standards with or without PHY. PHY at 50 mg/kg (p.o.) and all other standards exhibit significant (p < 0.05) anti-diarrheal effect in mice. The effect was prominent in the loperamide and propranolol groups. PHY co-treated with prazosin and propranolol was found to increase in latent periods along with a significant reduction in diarrheal section during the observation period than other individual or combined groups. Furthermore, molecular docking studies also suggested that PHY showed better interactions with the α- and β-adrenergic receptors, especially with α-ADR1a and β-ADR1. In the former case, PHY showed interaction with hydroxyl group of Ser192 at a distance of 2.91Å, while in the latter it showed hydrogen bond interactions with Thr170 and Lys297 with a distance of 2.65 and 2.72Å, respectively. PHY exerted significant anti-diarrheal effect in Swiss mice, possibly through blocking α- and β-adrenergic receptors.
The structure of a novel psychrophilic β-mannanase enzyme from Glaciozyma antarctica PI12 yeast has been modelled and analysed in detail. To our knowledge, this is the first attempt to model a psychrophilic β-mannanase from yeast. To this end, a 3D structure of the enzyme was first predicted using a threading method because of the low sequence identity (<30%) using MODELLER9v12 and simulated using GROMACS at varying low temperatures for structure refinement. Comparisons with mesophilic and thermophilic mannanases revealed that the psychrophilic mannanase contains longer loops and shorter helices, increases in the number of aromatic and hydrophobic residues, reductions in the number of hydrogen bonds and salt bridges and numerous amino acid substitutions on the surface that increased the flexibility and its efficiency for catalytic reactions at low temperatures.
The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.
The surface antigen (HBsAg) of hepatitis B virus (HBV) is highly conformational and generally evokes protective humoral immune response in human. A disulfide constrained random heptapeptide library displayed on the coat protein III of filamentous bacteriophage M13 was employed to select specific ligands that interact with HBsAg subtype ad. Fusion phages carrying the amino acid sequence ETGAKPH and other related sequences were isolated. The binding site of peptide ETGAKPH was located on the immunodominant region of HBsAg. An equilibrium binding assay in solution showed that the phage binds tightly to HBsAg with a relative dissociation constant (KDrel) of 2.9+/-0.9 nM. The phage bearing this peptide has the potential to be used as a diagnostic reagent and two assays for detecting HBsAg in blood samples are described.
Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.
The deduced amino acid sequences of segment A and B of two very virulent Infectious bursal disease virus (vvIBDV) isolates, UPM94/273 and UPM97/61 were compared with 25 other IBDV strains. Twenty amino acid residues (8 in VP1, 5 in VP2, 2 in VP3, 4 in VP4, 1 in VP5) that were common to vvIBDV strains were detected. However, UPM94/273 is an exceptional vvIBDV with usual amino acid substitutions. The differences in the divergence of segment A and B indicated that the vvIBDV strains may have been derived from genetic reassortment of a single ancestral virus or both segments have different ability to undergo genetic variation due to their different functional constraints.
We have carried out the synthesis of new 4-oxoquinazolin-3(4H)-yl)furan-2-carboxamide derivatives by the reaction between isatoic anhydride, 2-furoic hydrazide and substituted salicylaldehydes in ethanol: water (5:5 v/v) solvent system using p-TSA as a catalyst under ultrasound irradiation at room temperature. The structures of newly synthesized compounds were confirmed through spectral techniques such as IR, 1H NMR, 13C NMR, and LCMS. The important features of this protocol include simple and easy workup procedure, reaction carried out at ambient temperature, use of ultrasound and high yield of oxoquinazolin-3(4H)-yl)furan-2-carboxamides in short reaction time. The synthesized compounds 4a-4j were screened against tyrosinase enzyme and all these compounds found to be potent inhibitors with much lower IC50 value of 0.028 ± 0.016 to 1.775 ± 0.947 µM than the standard kojic acid (16.832 ± 1.162 µM). The kinetics mechanism for compound 4e was analyzed by Lineweaver-Burk plots which revealed that compound inhibited tyrosinase non-competitively by forming an enzyme-inhibitor complex. Along with this all the synthesized compounds (4a-4j) were scanned for their DPPH free radical scavenging ability. The outputs received through in vitro and in silico analysis are coherent to the each other with good binding energy values (kcal/mol) posed by synthesized ligands.
Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.
Hepatitis B virus surface mutants are of enormous importance because they are capable of escaping detection by serology and can infect both vaccinated and unvaccinated populations, thus putting the whole population at risk. This study aimed to detect and characterise hepatitis B-escaped mutants among blood donors and vaccinees. One thousand serum samples were collected for this study from blood donors and vaccinees. Hepatitis B surface antigen, antibodies and core antibodies were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. DNA detection was performed via nested polymerase chain reaction (PCR), and the S gene was sequenced and analysed using bioinformatics. Of the 1,000 samples that were screened, 5.5% (55/1,000) were found to be HBsAg-negative and anti-HBc- and HBV DNA-positive. All 55 isolates were found to belong to genotype B. Several mutations were found across all the sequences from synonymous and non-synonymous mutations, with the most nucleotide mutations occurring at position 342, where adenine was replaced by guanine, and cytosine at position 46 was replaced by adenine in 96.4% and 98% of the isolates, respectively. Mutation at position 16 of the amino acid sequence was found to be common to all the Malaysian isolates, with 85.7% of the mutations occurring outside the major hydrophilic region. This study revealed a prevalence of 5.5% for hepatitis B-escaped mutations among blood donors and vaccinated undergraduates, with the most common mutation being found at position 16, where glutamine was substituted with lysine.
Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.
Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs.
Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.
Bacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures.