Displaying publications 1 - 20 of 97 in total

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  1. Discovery of azetidine based ene-amides as potent bacterial enoyl ACP reductase (FabI) inhibitors
    Takhi M, Sreenivas K, Reddy CK, Munikumar M, Praveena K, Sudheer P, et al.
    Eur J Med Chem, 2014 Sep 12;84:382-94.
    PMID: 25036796 DOI: 10.1016/j.ejmech.2014.07.036
    A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are responsible for significant morbidity and mortality in community as well as hospital settings. The binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver microsomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated in a lethal murine systemic infection model.
  2. Fulltext Boesenbergia rotunda: From Ethnomedicine to Drug Discovery
    Eng-Chong T, Yean-Kee L, Chin-Fei C, Choon-Han H, Sher-Ming W, Li-Ping CT, et al.
    Evid Based Complement Alternat Med, 2012;2012:473637.
    PMID: 23243448 DOI: 10.1155/2012/473637
    Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.
  3. Fulltext Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling
    Hariono M, Choi SB, Roslim RF, Nawi MS, Tan ML, Kamarulzaman EE, et al.
    PLoS One, 2019;14(1):e0210869.
    PMID: 30677071 DOI: 10.1371/journal.pone.0210869
    Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 μM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 μM and 16 μM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.
  4. Fulltext Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia
    Yusof R, Ahmed MA, Jelip J, Ngian HU, Mustakim S, Hussin HM, et al.
    Emerg Infect Dis, 2016 Aug;22(8):1371-80.
    PMID: 27433965 DOI: 10.3201/eid2208.151885
    Infections of humans with the zoonotic simian malaria parasite Plasmodium knowlesi occur throughout Southeast Asia, although most cases have occurred in Malaysia, where P. knowlesi is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 P. knowlesi genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct P. knowlesi types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia.
  5. Polymethacrylate coated electrospun PHB fibers: An exquisite outlook for fabrication of paper-based biosensors
    Hosseini S, Azari P, Farahmand E, Gan SN, Rothan HA, Yusof R, et al.
    Biosens Bioelectron, 2015 Jul 15;69:257-64.
    PMID: 25765434 DOI: 10.1016/j.bios.2015.02.034
    Electrospun polyhydroxybutyrate (PHB) fibers were dip-coated by polymethyl methacrylate-co-methacrylic acid, poly(MMA-co-MAA), which was synthesized in different molar ratios of the monomers via free-radical polymerization. Fabricated platfrom was employed for immobilization of the dengue antibody and subsequent detection of dengue enveloped virus in enzyme-linked immunosorbent assay (ELISA). There is a major advantage for combination of electrospun fibers and copolymers. Fiber structre of electrospun PHB provides large specific surface area available for biomolecular interaction. In addition, polymer coated parts of the platform inherited the premanent presence of surface carboxyl (-COOH) groups from MAA segments of the copolymer which can be effectively used for covalent and physical protein immobilization. By tuning the concentration of MAA monomers in polymerization reaction the concentration of surface -COOH groups can be carefully controlled. Therefore two different techniques have been used for immobilization of the dengue antibody aimed for dengue detection: physical attachment of dengue antibodies to the surface and covalent immobilization of antibodies through carbodiimide chemistry. In that perspective, several different characterization techniques were employed to investigate the new polymeric fiber platform such as scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA) measurement and UV-vis titration. Regardless of the immobilization techniques, substantially higher signal intensity was recorded from developed platform in comparison to the conventional ELISA assay.
  6. Fulltext High proportion of knowlesi malaria in recent malaria cases in Malaysia
    Yusof R, Lau YL, Mahmud R, Fong MY, Jelip J, Ngian HU, et al.
    Malar J, 2014;13:168.
    PMID: 24886266 DOI: 10.1186/1475-2875-13-168
    Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.
  7. Fulltext Microsphere integrated microfluidic disk: synergy of two techniques for rapid and ultrasensitive dengue detection
    Hosseini S, Aeinehvand MM, Uddin SM, Benzina A, Rothan HA, Yusof R, et al.
    Sci Rep, 2015;5:16485.
    PMID: 26548806 DOI: 10.1038/srep16485
    The application of microfluidic devices in diagnostic systems is well-established in contemporary research. Large specific surface area of microspheres, on the other hand, has secured an important position for their use in bioanalytical assays. Herein, we report a combination of microspheres and microfluidic disk in a unique hybrid platform for highly sensitive and selective detection of dengue virus. Surface engineered polymethacrylate microspheres with carefully designed functional groups facilitate biorecognition in a multitude manner. In order to maximize the utility of the microspheres' specific surface area in biomolecular interaction, the microfluidic disk was equipped with a micromixing system. The mixing mechanism (microballoon mixing) enhances the number of molecular encounters between spheres and target analyte by accessing the entire sample volume more effectively, which subsequently results in signal amplification. Significant reduction of incubation time along with considerable lower detection limits were the prime motivations for the integration of microspheres inside the microfluidic disk. Lengthy incubations of routine analytical assays were reduced from 2 hours to 5 minutes while developed system successfully detected a few units of dengue virus. Obtained results make this hybrid microsphere-microfluidic approach to dengue detection a promising avenue for early detection of this fatal illness.
  8. Fulltext Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics
    Rothan HA, Ambikabothy J, Abdulrahman AY, Bahrani H, Golpich M, Amini E, et al.
    PLoS One, 2015;10(9):e0139248.
    PMID: 26418816 DOI: 10.1371/journal.pone.0139248
    The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.
  9. Mefenamic acid in combination with ribavirin shows significant effects in reducing chikungunya virus infection in vitro and in vivo
    Rothan HA, Bahrani H, Abdulrahman AY, Mohamed Z, Teoh TC, Othman S, et al.
    Antiviral Res, 2016 Mar;127:50-6.
    PMID: 26794398 DOI: 10.1016/j.antiviral.2016.01.006
    Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation of the viral vectors, Aedes aegypti and Aedes albopictus mosquitoes. Therefore, the absence of effective anti-CHIKV drugs to combat chikungunya outbreaks often leads to a significant impact on public health care. In this study, we investigated the antiviral activity of drugs that are used to alleviate infection symptoms, namely, the non-steroidal anti-inflammatory drugs (NSAIDs), on the premise that active compounds with potential antiviral and anti-inflammatory activities could be directly subjected for human use to treat CHIKV infections. Amongst the various NSAID compounds, Mefenamic acid (MEFE) and Meclofenamic acid (MECLO) showed considerable antiviral activity against viral replication individually or in combination with the common antiviral drug, Ribavirin (RIBA). The 50% effective concentration (EC50) was estimated to be 13 μM for MEFE, 18 μM for MECLO and 10 μM for RIBA, while MEFE + RIBA (1:1) exhibited an EC50 of 3 μM, and MECLO + RIBA (1:1) was 5 μM. Because MEFE is commercially available and its synthesis is easier compared with MECLO, MEFE was selected for further in vivo antiviral activity analysis. Treatment with MEFE + RIBA resulted in a significant reduction of hypertrophic effects by CHIKV on the mouse liver and spleen. Viral titre quantification in the blood of CHIKV-infected mice through the plaque formation assay revealed that treatment with MEFE + RIBA exhibited a 6.5-fold reduction compared with untreated controls. In conclusion, our study demonstrated that MEFE in combination with RIBA exhibited significant anti-CHIKV activity by impairing viral replication in vitro and in vivo. Indeed, this finding may lead to an even broader application of these combinatorial treatments against other viral infections.
  10. Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay
    Baharuddin A, Amir Hassan A, Othman R, Xu Y, Huang M, Ario Tejo B, et al.
    Chem Pharm Bull (Tokyo), 2014;62(10):947-55.
    PMID: 25273053
    In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
  11. Conformational and energy evaluations of novel peptides binding to dengue virus envelope protein
    Amir-Hassan A, Lee VS, Baharuddin A, Othman S, Xu Y, Huang M, et al.
    J Mol Graph Model, 2017 06;74:273-287.
    PMID: 28458006 DOI: 10.1016/j.jmgm.2017.03.010
    Effective novel peptide inhibitors which targeted the domain III of the dengue envelope (E) protein by blocking dengue virus (DENV) entry into target cells, were identified. The binding affinities of these peptides towards E-protein were evaluated by using a combination of docking and explicit solvent molecular dynamics (MD) simulation methods. The interactions of these complexes were further investigated by using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) and Molecular Mechanics Generalized Born Surface Area (MMGBSA) methods. Free energy calculations of the peptides interacting with the E-protein demonstrated that van der Waals (vdW) and electrostatic interactions were the main driving forces stabilizing the complexes. Interestingly, calculated binding free energies showed good agreement with the experimental dissociation constant (Kd) values. Our results also demonstrated that specific residues might play a crucial role in the effective binding interactions. Thus, this study has demonstrated that a combination of docking and molecular dynamics simulations can accelerate the identification process of peptides as potential inhibitors of dengue virus entry into host cells.
  12. Fulltext Small molecule grp94 inhibitors block dengue and Zika virus replication
    Rothan HA, Zhong Y, Sanborn MA, Teoh TC, Ruan J, Yusof R, et al.
    Antiviral Res, 2019 11;171:104590.
    PMID: 31421166 DOI: 10.1016/j.antiviral.2019.104590
    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.
  13. Discovery of Dengue Virus Inhibitors
    Abdullah AA, Lee YK, Chin SP, Lim SK, Lee VS, Othman R, et al.
    Curr Med Chem, 2020;27(30):4945-5036.
    PMID: 30514185 DOI: 10.2174/0929867326666181204155336
    To date, there is still no approved anti-dengue agent to treat dengue infection in the market. Although the only licensed dengue vaccine, Dengvaxia is available, its protective efficacy against serotypes 1 and 2 of dengue virus was reported to be lower than serotypes 3 and 4. Moreover, according to WHO, the risk of being hospitalized and having severe dengue increased in seronegative individuals after they received Dengvaxia vaccination. Nevertheless, various studies had been carried out in search of dengue virus inhibitors. These studies focused on the structural (C, prM, E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) of dengue virus as well as host factors as drug targets. Hence, this article provides an overall up-to-date review of the discovery of dengue virus inhibitors that are only targeting the structural and non-structural viral proteins as drug targets.
  14. Fulltext Iodine status among pregnant women in rural Sabah, Malaysia
    Lim KK, Chan YY, Teh CH, Ismail H, Yusof R, Muhi J, et al.
    Asia Pac J Clin Nutr, 2017 8 15;26(5):861-866.
    PMID: 28802296 DOI: 10.6133/apjcn.092016.06
    BACKGROUND AND OBJECTIVES: In 2000, legislation on mandatory universal salt iodisation was enacted in Sabah, Malaysia, to reduce the incidence of iodine deficiency disorders among its population. To evaluate the iodine levels among pregnant women from selected rural divisions in Sabah 13 years after the enactment of the universal salt iodisation programme.

    METHODS AND STUDY DESIGN: This cross-sectional study was conducted from 1 May to 30 June, 2013, in three rural divisions of Sabah (the Interior, the West Coast, and Kudat). Data regarding domestic iodised salt use and iodine-containing supplement consumption were obtained from respondents through face-to-face interviews; goitre enlargement was examined through palpation and graded according to the World Health Organization classification. Spot urine samples were also obtained to assess urinary iodine levels by using an in-house modified micromethod.

    RESULTS: In total, 534 pregnant women participated. The prevalence of goitre was 1.0% (n=5), noted only in the West Coast and Kudat divisions. Although all pregnant women consumed iodised salt, overall median urinary iodine concentration was only 106 μg/L, indicating insufficient iodine intake, with nearly two-thirds of the women (60%) having a median urinary iodine concentrations of <150 μg/L.

    CONCLUSIONS: Pregnant women from the rural divisions in Sabah still exhibit iodine deficiency disorder despite the mandatory universal salt iodisation programme. Iodine supplementation programmes targeting pregnant women are warranted.

  15. Fulltext Development of a passive liquid valve (PLV) utilizing a pressure equilibrium phenomenon on the centrifugal microfluidic platform
    Al-Faqheri W, Ibrahim F, Thio TH, Bahari N, Arof H, Rothan HA, et al.
    Sensors (Basel), 2015 Feb 25;15(3):4658-76.
    PMID: 25723143 DOI: 10.3390/s150304658
    In this paper, we propose an easy-to-implement passive liquid valve (PLV) for the microfluidic compact-disc (CD). This valve can be implemented by introducing venting chambers to control the air flow of the source and destination chambers. The PLV mechanism is based on equalizing the main forces acting on the microfluidic CD (i.e., the centrifugal and capillary forces) to control the burst frequency of the source chamber liquid. For a better understanding of the physics behind the proposed PLV, an analytical model is described. Moreover, three parameters that control the effectiveness of the proposed valve, i.e., the liquid height, liquid density, and venting chamber position with respect to the CD center, are tested experimentally. To demonstrate the ability of the proposed PLV valve, microfluidic liquid switching and liquid metering are performed. In addition, a Bradford assay is performed to measure the protein concentration and evaluated in comparison to the benchtop procedure. The result shows that the proposed valve can be implemented in any microfluidic process that requires simplicity and accuracy. Moreover, the developed valve increases the flexibility of the centrifugal CD platform for passive control of the liquid flow without the need for an external force or trigger.
  16. Association of dietary patterns with sociodemographic and health-related factors among coronary artery disease (CAD) patients
    Esmaili H, Mohd Yusof R, Abu Saad H, Ghaemian A, Darani Zad N
    Ecol Food Nutr, 2015;54(1):4-19.
    PMID: 25347717 DOI: 10.1080/03670244.2014.930031
    This study aimed to identify the association of dietary patterns with sociodemographic and health-related characteristics among coronary artery disease patients. In this cross-sectional study, the participants were 250 patients coronary artery disease aged ≥ 40 years old. Data collection was done using questionnaires related to sociodemographics, health-related factors, and food-frequency intake information. Three dietary patterns (traditional, western, and healthy) were obtained using principal component analysis. The result showed that dietary patterns were associated with sociodemographic and health-related factors. According to the result, all the factors were taken very seriously when planning a promotional program for healthy lifestyle in prevention of CAD.
  17. Biosensing enhancement of dengue virus using microballoon mixers on centrifugal microfluidic platforms
    Aeinehvand MM, Ibrahim F, Harun SW, Djordjevic I, Hosseini S, Rothan HA, et al.
    Biosens Bioelectron, 2015 May 15;67:424-30.
    PMID: 25220800 DOI: 10.1016/j.bios.2014.08.076
    Dengue is the current leading cause of death among children in several Latin American and Asian countries. Due to poverty in areas where the disease is prevalent and the high cost of conventional diagnostic systems, low cost devices are needed to reduce the burden caused by dengue infection. Centrifugal microfluidic platforms are an alternative solution to reduce costs and increase the availability of a rapid diagnostic system. The rate of chemical reactions in such devices often depends on the efficiency of the mixing techniques employed in their microfluidic networks. This paper introduces a micromixer that operates by the expansion and contraction of a microballoon to produce a consistent periodical 3D reciprocating flow. We established that microballoons reduced mixing time of 12 μl liquids from 170 min, for diffusional mixing, to less than 23 s. We have also tested the effect of the microballoon mixers on the detection of the dengue virus. The results indicate that employing a microballoon mixer enhances the detection sensitivity of the dengue virus by nearly one order of magnitude compared to the conventional ELISA method.
  18. Inhibitory effects of a peptide-fusion protein (Latarcin-PAP1-Thanatin) against chikungunya virus
    Rothan HA, Bahrani H, Shankar EM, Rahman NA, Yusof R
    Antiviral Res, 2014 Aug;108:173-80.
    PMID: 24929084 DOI: 10.1016/j.antiviral.2014.05.019
    Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
  19. Fulltext Three-dimensional culture environment increases the efficacy of platelet rich plasma releasate in prompting skin fibroblast differentiation and extracellular matrix formation
    Rothan HA, Djordjevic I, Bahrani H, Paydar M, Ibrahim F, Abd Rahmanh N, et al.
    Int J Med Sci, 2014;11(10):1029-38.
    PMID: 25136258 DOI: 10.7150/ijms.8895
    Platelet rich plasma clot- releasate (PRCR) shows significant influence on tissue regeneration in clinical trials. Although, the mechanism of PRCR effect on fibroblast differentiation has been studied on 2D culture system, a detailed investigation is needed to establish the role of PRCR in cell seeded in 3D scaffolds. Therefore, a study was conducted to evaluate the influence of PRCR in fibroblasts (DFB) differentiation and extracellular matrix formation on both 3D and 2D culture systems. Cell viability was measured using MTT assay and DFB differentiation was evaluated by determining the expression levels of nucleostamin and alpha smooth muscle actin (α-SMA), using indirect immunostaining and Western blotting. The expression levels of extracellular matrix genes (collagen-I, collagen-III, fibronectin and laminin) and focal adhesion formation gene (integrin beta-1) were measured using Real-time PCR. The PRCR at 10% showed significant effect on cells viability compared with 5% and 20% in both culture environments. The decrease in the expression levels of nucleostamin and the increase in α-SMA signify the DFB differentiation to myofibroblast-like cells that was prominently greater in 3D compared to 2D culture. In 3D culture systems, the total collage production, expression levels of the extracellular matrix gene and the focal adhesion gene were increased significantly compared to 2D culture. In conclusion, 3D culture environments enhances the proliferative and differentiation effects of PRCR on DFB, thereby potentially increases the efficacy of DFB for future tissue engineering clinical application.
  20. Fulltext Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide
    Rothan HA, Mohamed Z, Suhaeb AM, Rahman NA, Yusof R
    OMICS, 2013 Nov;17(11):560-7.
    PMID: 24044366 DOI: 10.1089/omi.2013.0056
    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
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