• Journal of Veterinary Science
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• v.19 no.6
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• pp.771-781
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• 2018

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

• Genomics & Informatics
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• v.19 no.4
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• pp.47.1-47.12
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• 2021

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• Journal of the Korean Chemical Society
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• v.67 no.1
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• pp.19-27
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• 2023

During the past few decades, toxicity science is shifting from observative to predictive science. New approach methodologies (NAMs), including in chemico, in silico, in vitro approach, gain attention to reduce, refine, replace the whole animal toxicity testing. However, actual acceptances of NAMs in regulatory decision-making have been limited due to low confidence. To address the current constraints, Accelerating the Pace of Chemical Risk Assessment (APCRA) initiative conducted several case studies and presented the perspectives of next generation risk assessment (NGRA). In this review, we suggested a concept and perspectives of NGRA through analysis on APCRA case studies.

• Natural Product Sciences
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• v.28 no.2
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• pp.45-52
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• 2022

The identification of Plasmodium falciparum enoyl acyl-carrier protein reductase (pfENR) is considered as a potential biological target against malaria. Trema orientalis is considered a rich source of phytochemicals useful in malaria treatment. This study evaluated the in-vitro inhibitory activity of the extract and isolated compounds of T. orientalis leaf; the isolated compounds and the analogues of the most active compound were subjected to in-silico molecular docking studies on pfENR. The methanolic extract of T. orientalis was subjected to repeated chromatographic separation which led to the isolation of some compounds. The isolated compounds from the plant were examined for their antimalarial activity using β-hematin inhibition assay. Virtual screening via molecular docking and ADMET studies were conducted to gain insight into the mechanism of binding of ligand and to identify effective pfENR inhibitors. The isolated compounds and the analogues of the most active isolates were gotten from PubChem library for use in docking study. Hexacosanol and β-sitosterol showed inhibition of the β-hematin formation. The docking results showed that hexacosanol, β-sitosterol and the analogues of β-sitosterol displayed binding energy ranging between -6.1 kcal/mol and -11.6 kcal/mol. Sitosterol glucoside has the highest docking score. Some of the ligands showed more binding affinity than known bioactive compounds used as reference. Analogues of β-sitosterol has been shown to be potential inhibitors of pfENR, therefore, the findings from this study suggest that sitosterol glucoside and ergosterol peroxide could act as antimalarial agents after further lead optimisation investigations.

• Genomics & Informatics
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• v.21 no.1
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• pp.3.1-3.10
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• 2023

Characterization as well as prediction of the secondary and tertiary structure of hypothetical proteins from their amino acid sequences uploaded in databases by in silico approach are the critical issues in computational biology. Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), which is responsible for pneumonia alike diseases, possesses a wide range of proteins of which many are still uncharacterized. The current study was conducted to reveal the physicochemical characteristics and structures of an uncharacterized protein Q6S8D9_SARS of SARS-CoV. Following the common flowchart of characterizing a hypothetical protein, several sophisticated computerized tools e.g., ExPASy Protparam, CD Search, SOPMA, PSIPRED, HHpred, etc. were employed to discover the functions and structures of Q6S8D9_SARS. After delineating the secondary and tertiary structures of the protein, some quality evaluating tools e.g., PROCHECK, ProSA-web etc. were performed to assess the structures and later the active site was identified also by CASTp v.3.0. The protein contains more negatively charged residues than positively charged residues and a high aliphatic index value which make the protein more stable. The 2D and 3D structures modeled by several bioinformatics tools ensured that the proteins had domain in it which indicated it was functional protein having the ability to trouble host antiviral inflammatory cytokine and interferon production pathways. Moreover, active site was found in the protein where ligand could bind. The study was aimed to unveil the features and structures of an uncharacterized protein of SARS-CoV which can be a therapeutic target for development of vaccines against the virus. Further research are needed to accomplish the task.

• Genomics & Informatics
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• v.21 no.3
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.871-879
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• 2024

Our group had isolated Bifidobacterium breve strain BS2-PB3 from human breast milk. In this study, we sequenced the whole genome of B. breve BS2-PB3, and with a focus on its safety profile, various probiotic characteristics (presence of antibiotic resistance genes, virulence factors, and mobile elements) were then determined through bioinformatic analyses. The antibiotic resistance profile of B. breve BS2-PB3 was also evaluated. The whole genome of B. breve BS2-PB3 consisted of 2,268,931 base pairs with a G-C content of 58.89% and 2,108 coding regions. The average nucleotide identity and whole-genome phylogenetic analyses supported the classification of B. breve BS2-PB3. According to our in silico assessment, B. breve BS2-PB3 possesses antioxidant and immunomodulation properties in addition to various genes related to the probiotic properties of heat, cold, and acid stress, bile tolerance, and adhesion. Antibiotic susceptibility was evaluated using the Kirby-Bauer disk-diffusion test, in which the minimum inhibitory concentrations for selected antibiotics were subsequently tested using the Epsilometer test. B. breve BS2-PB3 only exhibited selected resistance phenotypes, i.e., to mupirocin (minimum inhibitory concentration/MIC >1,024 ㎍/ml), sulfamethoxazole (MIC>1,024 ㎍/ml), and oxacillin (MIC >3 ㎍/ml). The resistance genes against those antibiotics, i.e., ileS, mupB, sul4, mecC and ramA, were detected within its genome as well. While no virulence factor was detected, four insertion sequences were identified within the genome but were located away from the identified antibiotic resistance genes. In conclusion, B. breve BS2-PB3 demonstrated a sufficient safety profile, making it a promising candidate for further development as a potential functional food.

• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.552-560
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• 2020

Human carbonic anhydrase (CA) isozyme II has been used as protein target for disorder treatment including glaucoma. Current clinically used sulfonamide-based CA inhibitors can induce side effects, and so alternatives are required. This study aimed to investigate a natural CA inhibitor from Murraya paniculata. The previously developed yeast-based assay was used to screen 14 compounds isolated from M. paniculata and identified by NMR analysis for anti-human CA isozyme II (hCAII) activity. Cytotoxicity of the compounds was also tested using the same yeast-based assay but in a different cultivation condition. Two flavonoid candidate compounds, 5, 6, 7, 8, 3', 4', 5'-heptamethoxyflavone (4) and 3, 5, 7, 8, 3', 4', 5'-heptamethoxyflavone (9), showed potent inhibitory activity against hCAII with a minimal effective concentration of 10.8 and 21.5 μM, respectively, while they both exhibited no cytotoxic effect, even at the highest concentration tested (170 μM). The results from an in vitro esterase assay of the two candidates confirmed their hCAII inhibitory activity with IC₅₀ values of 24.0 and 34.3 μM, respectively. To investigate the potential inhibition mechanism of compound 4, in silico molecular docking was performed using the FlexX and SwissDock software. This revealed that compound 4 coordinated with the Zn²⁺ ion in the hCAII active site through its methoxy oxygen at a distance of 1.60 Å (FlexX) or 2.29 Å (SwissDock). The interaction energy of compound 4 with hCAII was -13.36 kcal/mol. Thus, compound 4 is a potent novel flavonoid-based hCAII inhibitor and may be useful for further anti-CAII design and development.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7473-7482
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• 2013

The G-protein coupled receptor 87 (GPR87) is a recently discovered orphan GPCR which means that the search of their endogenous ligands has been a novel challenge. GPR87 has been shown to be overexpressed in squamous cell carcinomas (SCCs) or adenocarcinomas in lungs and bladder. The 3D structure of GPR87 was here modeled using two templates (2VT4 and 2ZIY) by a threading method. Functional assignment of GPR87 by SVM revealed that along with transporter activity, various novel functions were predicted. The 3D structure was further validated by comparison with structural features of the templates through Verify-3D, ProSA and ERRAT for determining correct stereochemical parameters. The resulting model was evaluated by Ramachandran plot and good 3D structure compatibility was evidenced by DOPE score. Molecular dynamics simulation and solvation of protein were studied through explicit spherical boundaries with a harmonic restraint membrane water system. A DRY-motif (Asp-Arg-Tyr sequence) was found at the end of transmembrane helix3, where GPCR binds and thus activation of signals is transduced. In a search for better inhibitors of GPR87, in silico modification of some substrate ligands was carried out to form polar interactions with Arg115 and Lys296. Thus, this study provides early insights into the structure of a major drug target for SCCs.