28 research outputs found
Homology Modeling of Coagulase in Staphylococcus aureus
The close correlation between the ability of coagulase to clot blood plasma and their capacity to produce disease, and the
corresponding absence of this property in nonpathogenic strains, have led to the assumption that the coagulase, plays important
role in the pathogenesis of disease. Currently, crystal structure of coagulase in Staphylococcus aureus remains indefinable. Thus, the
objectives of this research is to generate the three dimensional model of coagulase in S. aureus by using homology modeling
approach. In this study, we used bioinformatics tools and databases such as BLAST (Basic Local Alignment Search Tool), GenBank,
PDB (Protein Databank), and Discovery Studio to gain specific functional insights into coagulase. The model was validated using
protein structure checking tools such as PROCHECK, Verify 3D and CE (Combinatorial Extension) for reliability. Therefore,
structure prediction of coagulase in S. aureus can provide preliminary knowledge for understanding the function of the protein.
The information from this finding will provide important information into the action and regulation mechanism of the coagulase
protein in S. aureus
Protein expression of Myt272-3 recombinant clone and in silico prediction of a possible vaccine candidate against Mycobacterium tuberculosis
Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis.Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation & Pulldown. Protein sequence was analysed in silico using bioinformatics software for the prediction of allergenicity, antigenicity, MHC-I and MHC-II binding, and B-cell epitope binding.Results: The candidate protein was a non-allergen with 15.19 % positive predictive value. It was also predicted to be antigenic, with binding affinity to MHC-I and MHC-II, as well as B-cell epitope binding.Conclusion: The predicted results obtained in this study provide a guide for practical design of a new tuberculosis vaccine.Keywords: Vaccine, Mycobacterium tuberculosis, Epitope prediction, Histocompatibility complexe
Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli
AbstractBackgroundBacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12±0.01μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain.ResultsThe endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51±0.02μmol/min/mL) compared with the cell-bound fraction (0.37±0.02μmol/min/mL) and the extracellular fraction (0.33±0.01μmol/min/mL). The recombinant endoglucanase was approximately 56kDa, with optimal enzyme activity at 60°C and pH6.0. The activity of the enzyme was enhanced by the addition of Ca2+. However, the enzyme was inhibited by other metal ions in the following order: Fe3+>Ni2+>Cu2+>Mn2+=Zn2+>Mg2+>Cd2+>Cr2+. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside.ConclusionsThe recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose
Vaccine research and development: tuberculosis as a global health threat
One of the aims of the World Health Organisation (WHO) Millennium Development Goals (MDG) is to reduce the number of cases of tuberculosis (TB) infection by the year 2015. However, 9 million new cases were reported in 2013, with an estimated 480,000 new cases of multi-drug resistant tuberculosis (MDR-TB) globally. Bacille Calmette-Guérin (BCG) is the most available and currently used candidate vaccine against tuberculosis; it prevents childhood TB, but its effectiveness against pulmonary TB in adults and adolescents is disputed. To achieve the goal of the WHO MDG, the need for a new improved vaccine is of primary importance. This review highlights several articles that have reported vaccine development. There are about 16 TB vaccines in different phases of clinical trials at the time of writing, which include recombinant peptide/protein, live-attenuated and recombinant live-attenuated, protein/adjuvant, viral-vectored, and immunotherapeutic vaccine. Further studies in reverse vaccinology and massive campaigns on vaccination are needed in order to achieve the target for TB eradication by 2050
In vitro and in silico studies of chalcone synthase variant 2 in Boesenbergia rotunda and its substrate specificity
In this study, transformation of BrCHS var 2 into B. rotunda cell suspension culture, followed by chalcone synthase enzymatic assay and HPLC analysis was conducted to investigate whether the substrate specificity for BrCHS var 2 is either cinnamoyl-CoA or p-coumaroyl-CoA. The HPLC profile showed an increase in the amount of pinocembrin chalcone when cinnamoyl-CoA and malonyl-CoA were added but not p-coumaroyl-CoA. Molecular docking was performed to explore the binding of cinnamoyl-CoA and p-coumaroyl-CoA to BrCHS var 2 receptor and the docking results showed that cinnamoyl-CoA formed numerous hydrogen bonds and more negative docked energy than p-coumaroyl-CoA. Cinnamoyl-CoA showed good interactions with Cys 164 to initiate the subsequent formation of pinocembrin chalcone, whereas the hydroxyl group of p-coumaroyl-CoA formed an unfavorable interaction with Gln 161 that caused steric hindrance to subsequent formation of naringenin chalcone. Docked conformation analysis results also showed that malonyl-CoA formed hydrogen bonding with Cys 164, His 303, and Asn 336 residues in BrCHS var 2. The results show that cinnamoyl-CoA is the preferred substrate for BrCHS var 2
Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts
Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (−7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection
Novel Peptides Inhibit Zika NS2B-NS3 Serine Protease and Virus Replication in Human Hepatic Cell Line
Zika virus (ZIKV) is a Flavivirus associated with several neurological complications. Currently, there are no vaccines or cures available and an efficient antiviral treatment is urgently needed to combat ZIKV infection. Herein, we targeted ZIKV NS2B-NS3 serine protease with short peptides to inhibit ZIKV replication in human hepatic cell line (WRL-68). The short peptide inhibitors were designed using Hyperchem 8.0.10 software. Docking energy and binding configuration were calculated using HADDOCK webserver. ZIKV NS2B-NS3 protease was produced as a recombinant single peptide in Escherichia coli and the protease activity was examined by measuring the cleavage of a fluorescent substrate in the presence of the peptides or aprotinin as a standard protease inhibitor. Computational analysis revealed that the short peptides, AYA2 and AYA9, exhibited lower docking energy to ZIKV protease than aprotinin. Both peptides also possessed lower half maximal inhibitory concentration (IC50), 30.9 and 22.1 µM respectively, against ZIKV protease activity when compared to aprotinin (35.4 µM). Interestingly, AYA2 and AYA9 exhibited minimal cytotoxic effects in WRL-68 cells and showed considerable inhibition against ZIKV replication in vitro at half maximal effective concentration (EC50) of 40.73 ± 2.3 µM and 34.65 ± 1.8 µM respectively. Fusion of these two peptides to MAP30 peptide substantially reduced the IC50 of ZIKV protease inhibition to 1.1 µM and inhibited ZIKV replication at EC50 of 0.5157 ± 0.03 µM. In sum, we reported novel peptides that effectively inhibited ZIKV replication in vitro. This study represents a cost-effective strategy of developing peptide inhibitors by shortening the peptides and producing them in recombinant form. © 2019, Springer Nature B.V
Idala: An unnamed Function Peptide Vaccine for Tuberculosis
Purpose: To evaluate Myt272 protein antigenicity and immunogenicity by
trial vaccination in mice and its in silico analysis as a potential
peptide vaccine for tuberculosis. Methods: Myt272 gene, which has 100 %
identity with Mycobacterium tuberculosis H37Rv unknown function gene
Rv3424c, was ligated by genomic shotgun approach into the expression
vector pQE32, and transformed into Escherichia coli SG13009. Expression
during cell growth was induced by
isopropyl-β-D-thiogalactopyranoside. The recombinant protein was
isolated from the harvested cell lysate and injected in mice for
immunogenicity experiment up to 42 days. ELISA tests with anti-His
antibodies were performed on the collected individual blood
samples’ sera. Color development in a microplate reader was
measured at 450 nm. Results: The protein was predicted to have a mass
of approximately 13 kDa and was present in the soluble fraction of the
cell lysate. The immunogenicity test on Myt272 protein revealed very
statistically significant high levels of antibodies detected by ELISA
in the sera of immunized group of mice compared to negative controls.
Conclusion: A 10.1 kD unnamed function (IDALA) protein from Rv3424 gene
could be the potential peptide vaccine for tuberculosis tested by mice
immunogenicity experiment
Carnosine exhibits significant antiviral activity against Dengue and Zika virus
Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito-borne diseases whereas ZIKV infection occasionally re-emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β-alanyl-l-histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell-based assays were performed to validate the computational results. Mode-of-inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC50 values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode-of-inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV. © 2019 European Peptide Society and John Wiley & Sons, Ltd
Small molecule grp94 inhibitors block dengue and Zika virus replication
Two major flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), cause severe health and economic burdens worldwide. Recently, genome-wide screenings have uncovered the importance of regulators of the Hrd1 ubiquitin ligase-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) pathway for flavivirus replication in host cells. Here we report the identification of the compound Bardoxolone methyl (CDDO-me) as a potent inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD, which possesses a broad-spectrum activity against both DENV and ZIKV. Cellular thermal shift assay (CETSA) suggested that CDDO-me binds to grp94, a key component of the Hrd1 pathway, at a low nanomolar concentration, whereas interaction was not detected with its paralog Hsp90. CDDO-me and the grp94 inhibitor PU-WS13 substantially suppressed DENV2 replication and the cytopathic effects caused by DENV and ZIKV infection. The antiviral activities of both compounds were demonstrated for all four DENV serotypes and four ZIKV strains in multiple human cell lines. This study defines grp94 as a crucial host factor for flavivirus replication and identified CDDO-me as a potent small molecule inhibitor of flavivirus infection. Inhibition of grp94 may contribute to the antiviral activity of CDDO-me. Further investigation of grp94 inhibitors may lead to a new class of broad-spectrum anti-flaviviral medications. © 2019 Elsevier B.V