Rohana Yusof | University of Malaya, Malaysia (original) (raw)

Papers by Rohana Yusof

The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-enceph... more The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-encephalitogenic dengue 3 virus strains isolated in Malaysia were determined and compared. Multiple sequence alignment revealed a high degree of similarity in the E protein of the strains suggesting that neurovirulence of these four encephalitogenic strains is not attributed to this protein. (C) 2004 Royal society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Inorg Chem, 1993

An electrochemical approach to assessing the degree of electronic coupling in mixed-valence binuc... more An electrochemical approach to assessing the degree of electronic coupling in mixed-valence binuclear complexes is outlined. The method relies on the comparison of electrochemical potential shifts induced at both the directly and indirectly perturbed metal sites when a ligand substitution process is carried out at one site, e.g., [symmetric] ( N H~)~R U -L~~R U ( N H~)~~+ /~+ /~+ -[asymmetric] L(NH~)~RU-LI,,-R~(NH~)~~+/~+/~+, where the bridging ligand

Virology Journal, 2016

Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is i... more Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is infrequently mutated in human tumours. Its expression is posttranscriptionally regulated and largely G0 restricted. We have previously shown that E6/E7 oncoproteins encoded by human papillomavirus (HPV) type 16, which is a high-risk type for cervical cancer development, must target p130 to promote the host cell to exit from quiescence (G0) state and enter S phase of the cell cycle. P130 is associated with the DREAM (DP, RB-like, E2F and MuvB) complex in G0/G1, which prevents S phase progression by repressing transcription of E2F-regulated genes. E7 proteins could potentially disrupt the p130-DREAM complex through two known mechanisms: direct interaction with p130 or induction of cyclin dependent kinase 2 (CDK2) phosphorylation by interacting with its inhibitor, p21(CIP1). In this study we have used p130 mutants deficient in binding the E7 LXCXE domain (p130mE7), unphosphorylatable by CDK2 (p130PM22) or a combination of both (p130PM22/mE7) to investigate these mechanisms used by E7 proteins to disrupt the p130-DREAM complex and promote cell cycle progression. We found that HPV16 E7 binding to p130 through its LXCXE domain was absolutely required to disrupt p130-DREAM to promote S phase of the cell cycle, as HPV16 E7 was unable to suppress p130mE7 but could suppress p130PM22. In contrast, the E7 protein encoded by a cutaneous HPV type that lacks a functional LXCXE domain, HPV 48 E7, was also able to disrupt p130-DREAM to promote cell cycling, but through the alternative mechanism. Thus, HPV48 E7 could suppress a cell cycle block imposed by p130mE7, but was unable to suppress p130PM22. Overall, these results indicate that suppression of p130 is required for HPV-induced cell cycling, and that different HPV E7 proteins can use alternative mechanisms to achieve this.

Antiviral Research, 2016

Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation... more 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.

Scientific Reports, Nov 9, 2015

The application of microfluidic devices in diagnostic systems is well-established in contemporary... more 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.

ELECTROPHORESIS, 2015

Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a non-uniform ele... more Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a non-uniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Because cells of the same genotype in different physiological and pathological states have unique morphological and structural features, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift (CMIS) image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties. This article is protected by copyright. All rights reserved.

PLOS ONE, 2015

The production of short anticancer peptides in recombinant form is an alternative method for cost... more 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.

The crude extracts and the methanol and hexane partitioned fractions from the rhizomes of six Zin... more The crude extracts and the methanol and hexane partitioned fractions from the rhizomes of six Zingiberaceae comprising five Curcumas and one Zingiber were screened for dengue-2 (Den2) virus NS2B/NS3 protease inhibition. The percentage inhibition of Den2 virus NS2B/NS3 protease cleavage of the substrate showed linear dose-dependent increment for all the samples tested. The crude extracts were less potent than the best of the partitioned fractions. For all three concentrations studied, the methanol fractions of the extracts of Curcuma longa (L.) (CL), Zingiber zerumbet Smith (ZZ) and Curcuma rubescen Roxb. (CR) were much more inhibiting than the corresponding hexane fractions but the converse was true for Curcuma aeroginosa Roxb. (CA). However, the inhibiting activities of the two partitioned fractions of the extracts of Curcuma mangga Roxb. (CM) and Curcuma xanthorhiza Roxb. (CX) were found to be similar. The CL methanol fraction exhibited the strongest inhibitory activity (91.3±3.1%...

Applied Surface Science, 2015

ABSTRACT

Cell-penetrating peptides (CPPs) have shown great potential in ligand-mediated drug delivery systems

Lab Chip, 2015

Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel bioche... more Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.

PLOS ONE, 2015

The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the ... more The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the cause of numerous outbreaks in tropical and temperate regions, worldwide. There is limited understanding regarding the underlying molecular mechanisms involved in CHIKV replication and how the virus interacts with its host. In the present study, comparative proteomics was used to identify secreted host proteins that changed in abundance in response to early CHIKV infection. Two-dimensional gel electrophoresis was used to analyse and compare the secretome profiles of WRL-68 cells infected with CHIKV against mock control WRL-68 cells. The analysis identified 25 regulated proteins in CHIKV infected cells. STRING network analysis was then used to predict biological processes that may be affected by these proteins. The processes predicted to be affected include signal transduction, cellular component and extracellular matrix (ECM) organization, regulation of cytokine stimulus and immune response. These results provide an initial view of CHIKV may affect the secretome of infected cells during early infection. The results presented here will compliment earlier results from the study of late host response. However, functional characterization will be necessary to further enhance our understanding of the roles played by these proteins in the early stages of CHIKV infection in humans.

Acta Tropica, 2015

Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alter... more Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

PLOS ONE, 2015

Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have le... more Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.

Biochemical Engineering Journal, 2015

ABSTRACT Polymethylmethacrylate-co-methacrylic acid, poly(MMA-co-MAA) coatings were produced with... more ABSTRACT Polymethylmethacrylate-co-methacrylic acid, poly(MMA-co-MAA) coatings were produced with different initial molar ratios of monomers (MMA and MAA) in free-radical polymerization reaction. Polymeric platforms were specifically designed with controlled concentration of surface-exposed carboxyl (COOH) groups that can be used as a desirable functionality for protein immobilization. Spin-coated chips were used for antibody (Ab) immobilization in order to investigate the influence of COOH surface concentration on dengue virus detection efficiency in enzyme-linked immunosorbent assay (ELISA) experiment. Successful immobilization of Ab was achieved by two different techniques: (1) physical adsorption; and (2) covalent immobilization by carbodiimide coupling between the surface COOH groups and amine functionalities of dengue Ab molecules. Produced polymer coatings were characterized with surface spectroscopy techniques (Raman and X-ray photoelectron spectroscopy, XPS) and water-in-air contact angle (WCA) measurements. In particular, this research concentrated on the aging effect on the availability and activity of surface COOH groups. For that reason, WCA and Ab immobilization (ELISA) experiments were repeated on coated biochips after 3, 6 and 9 months of storage. Results in this paper describe the robust and sustainable functionalized polymeric platform that can be used effectively for protein activation and development of novel biosensors.

Dengue virus infects millions of people worldwide and there is no vaccine or antidengue therapeut... more Dengue virus infects millions of people worldwide and there is no vaccine or antidengue therapeutic available. Screening large numbers of medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds. Therefore, this study was designed to identify anti-dengue activities in nineteen medicinal plant extracts that are used in traditional medicine. Local medicinal plants Vernonia cinerea, Hemigraphis reptans, Hedyotis auricularia, Laurentia longiflora, Tridax procumbers and Senna angustifolia were used in this study. The highest inhibitory activates against dengue NS2B-NS3pro was observed in ethanolic extract of S. angustifolia leaves, methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems. These findings were further verified by in vitro viral inhibition assay. Methanolic extract of V. cinerea leaves, ethanol extract of T. procumbens stems and at less extent ethanolic extract of S. angustifolia leaves were able to maintain the normal morphology of DENV2-infected Vero cells without causing much cytopathic effects (CPE). The percentage of viral inhibition of V. cinerea and T. procumbens extracts were significantly higher than S. angustifolia extract as measured by plaque formation assay and RT-qPCR. In conclusion, The outcome of this study showed that the methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems possessed high inhibitory activates against dengue virus that worth more investigation.

BMC microbiology, Jan 31, 2014

Although there have been considerable advances in the study of dengue virus, no vaccines or anti-... more Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells. The Ltc 1 peptide showed a significantly inhibitory effect against the dengue protease NS2B-NS3pro at 37°C, a physiological human temperature, (IC50, 12.68 ± 3.2 μM), and greater inhibitory effect was observed at 40°C, a temperature similar to a high fever (IC50, 6.58 ± 4.1 μM). A greater reduction in viral load (p.f.u./ml) was observed at simultaneous (0.7 ± 0.3 vs. 7.2 ± 0.5 control) and post-treatment (1.8 ± 0.7 vs. 6.8 ± 0.6 control) co...

European Journal of Cancer, 2014

The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-enceph... more The amino acid sequences of the envelope (E) protein of four encephalitogenic and five non-encephalitogenic dengue 3 virus strains isolated in Malaysia were determined and compared. Multiple sequence alignment revealed a high degree of similarity in the E protein of the strains suggesting that neurovirulence of these four encephalitogenic strains is not attributed to this protein. (C) 2004 Royal society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Inorg Chem, 1993

An electrochemical approach to assessing the degree of electronic coupling in mixed-valence binuc... more An electrochemical approach to assessing the degree of electronic coupling in mixed-valence binuclear complexes is outlined. The method relies on the comparison of electrochemical potential shifts induced at both the directly and indirectly perturbed metal sites when a ligand substitution process is carried out at one site, e.g., [symmetric] ( N H~)~R U -L~~R U ( N H~)~~+ /~+ /~+ -[asymmetric] L(NH~)~RU-LI,,-R~(NH~)~~+/~+/~+, where the bridging ligand

Virology Journal, 2016

Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is i... more Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is infrequently mutated in human tumours. Its expression is posttranscriptionally regulated and largely G0 restricted. We have previously shown that E6/E7 oncoproteins encoded by human papillomavirus (HPV) type 16, which is a high-risk type for cervical cancer development, must target p130 to promote the host cell to exit from quiescence (G0) state and enter S phase of the cell cycle. P130 is associated with the DREAM (DP, RB-like, E2F and MuvB) complex in G0/G1, which prevents S phase progression by repressing transcription of E2F-regulated genes. E7 proteins could potentially disrupt the p130-DREAM complex through two known mechanisms: direct interaction with p130 or induction of cyclin dependent kinase 2 (CDK2) phosphorylation by interacting with its inhibitor, p21(CIP1). In this study we have used p130 mutants deficient in binding the E7 LXCXE domain (p130mE7), unphosphorylatable by CDK2 (p130PM22) or a combination of both (p130PM22/mE7) to investigate these mechanisms used by E7 proteins to disrupt the p130-DREAM complex and promote cell cycle progression. We found that HPV16 E7 binding to p130 through its LXCXE domain was absolutely required to disrupt p130-DREAM to promote S phase of the cell cycle, as HPV16 E7 was unable to suppress p130mE7 but could suppress p130PM22. In contrast, the E7 protein encoded by a cutaneous HPV type that lacks a functional LXCXE domain, HPV 48 E7, was also able to disrupt p130-DREAM to promote cell cycling, but through the alternative mechanism. Thus, HPV48 E7 could suppress a cell cycle block imposed by p130mE7, but was unable to suppress p130PM22. Overall, these results indicate that suppression of p130 is required for HPV-induced cell cycling, and that different HPV E7 proteins can use alternative mechanisms to achieve this.

Antiviral Research, 2016

Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation... more 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.

Scientific Reports, Nov 9, 2015

The application of microfluidic devices in diagnostic systems is well-established in contemporary... more 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.

ELECTROPHORESIS, 2015

Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a non-uniform ele... more Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a non-uniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Because cells of the same genotype in different physiological and pathological states have unique morphological and structural features, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift (CMIS) image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties. This article is protected by copyright. All rights reserved.

PLOS ONE, 2015

The production of short anticancer peptides in recombinant form is an alternative method for cost... more 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.

The crude extracts and the methanol and hexane partitioned fractions from the rhizomes of six Zin... more The crude extracts and the methanol and hexane partitioned fractions from the rhizomes of six Zingiberaceae comprising five Curcumas and one Zingiber were screened for dengue-2 (Den2) virus NS2B/NS3 protease inhibition. The percentage inhibition of Den2 virus NS2B/NS3 protease cleavage of the substrate showed linear dose-dependent increment for all the samples tested. The crude extracts were less potent than the best of the partitioned fractions. For all three concentrations studied, the methanol fractions of the extracts of Curcuma longa (L.) (CL), Zingiber zerumbet Smith (ZZ) and Curcuma rubescen Roxb. (CR) were much more inhibiting than the corresponding hexane fractions but the converse was true for Curcuma aeroginosa Roxb. (CA). However, the inhibiting activities of the two partitioned fractions of the extracts of Curcuma mangga Roxb. (CM) and Curcuma xanthorhiza Roxb. (CX) were found to be similar. The CL methanol fraction exhibited the strongest inhibitory activity (91.3±3.1%...

Applied Surface Science, 2015

ABSTRACT

Cell-penetrating peptides (CPPs) have shown great potential in ligand-mediated drug delivery systems

Lab Chip, 2015

Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel bioche... more Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.

PLOS ONE, 2015

The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the ... more The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the cause of numerous outbreaks in tropical and temperate regions, worldwide. There is limited understanding regarding the underlying molecular mechanisms involved in CHIKV replication and how the virus interacts with its host. In the present study, comparative proteomics was used to identify secreted host proteins that changed in abundance in response to early CHIKV infection. Two-dimensional gel electrophoresis was used to analyse and compare the secretome profiles of WRL-68 cells infected with CHIKV against mock control WRL-68 cells. The analysis identified 25 regulated proteins in CHIKV infected cells. STRING network analysis was then used to predict biological processes that may be affected by these proteins. The processes predicted to be affected include signal transduction, cellular component and extracellular matrix (ECM) organization, regulation of cytokine stimulus and immune response. These results provide an initial view of CHIKV may affect the secretome of infected cells during early infection. The results presented here will compliment earlier results from the study of late host response. However, functional characterization will be necessary to further enhance our understanding of the roles played by these proteins in the early stages of CHIKV infection in humans.

Acta Tropica, 2015

Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alter... more Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.

PLOS ONE, 2015

Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have le... more Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.

Biochemical Engineering Journal, 2015

ABSTRACT Polymethylmethacrylate-co-methacrylic acid, poly(MMA-co-MAA) coatings were produced with... more ABSTRACT Polymethylmethacrylate-co-methacrylic acid, poly(MMA-co-MAA) coatings were produced with different initial molar ratios of monomers (MMA and MAA) in free-radical polymerization reaction. Polymeric platforms were specifically designed with controlled concentration of surface-exposed carboxyl (COOH) groups that can be used as a desirable functionality for protein immobilization. Spin-coated chips were used for antibody (Ab) immobilization in order to investigate the influence of COOH surface concentration on dengue virus detection efficiency in enzyme-linked immunosorbent assay (ELISA) experiment. Successful immobilization of Ab was achieved by two different techniques: (1) physical adsorption; and (2) covalent immobilization by carbodiimide coupling between the surface COOH groups and amine functionalities of dengue Ab molecules. Produced polymer coatings were characterized with surface spectroscopy techniques (Raman and X-ray photoelectron spectroscopy, XPS) and water-in-air contact angle (WCA) measurements. In particular, this research concentrated on the aging effect on the availability and activity of surface COOH groups. For that reason, WCA and Ab immobilization (ELISA) experiments were repeated on coated biochips after 3, 6 and 9 months of storage. Results in this paper describe the robust and sustainable functionalized polymeric platform that can be used effectively for protein activation and development of novel biosensors.

Dengue virus infects millions of people worldwide and there is no vaccine or antidengue therapeut... more Dengue virus infects millions of people worldwide and there is no vaccine or antidengue therapeutic available. Screening large numbers of medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds. Therefore, this study was designed to identify anti-dengue activities in nineteen medicinal plant extracts that are used in traditional medicine. Local medicinal plants Vernonia cinerea, Hemigraphis reptans, Hedyotis auricularia, Laurentia longiflora, Tridax procumbers and Senna angustifolia were used in this study. The highest inhibitory activates against dengue NS2B-NS3pro was observed in ethanolic extract of S. angustifolia leaves, methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems. These findings were further verified by in vitro viral inhibition assay. Methanolic extract of V. cinerea leaves, ethanol extract of T. procumbens stems and at less extent ethanolic extract of S. angustifolia leaves were able to maintain the normal morphology of DENV2-infected Vero cells without causing much cytopathic effects (CPE). The percentage of viral inhibition of V. cinerea and T. procumbens extracts were significantly higher than S. angustifolia extract as measured by plaque formation assay and RT-qPCR. In conclusion, The outcome of this study showed that the methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems possessed high inhibitory activates against dengue virus that worth more investigation.

BMC microbiology, Jan 31, 2014

Although there have been considerable advances in the study of dengue virus, no vaccines or anti-... more Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells. The Ltc 1 peptide showed a significantly inhibitory effect against the dengue protease NS2B-NS3pro at 37°C, a physiological human temperature, (IC50, 12.68 ± 3.2 μM), and greater inhibitory effect was observed at 40°C, a temperature similar to a high fever (IC50, 6.58 ± 4.1 μM). A greater reduction in viral load (p.f.u./ml) was observed at simultaneous (0.7 ± 0.3 vs. 7.2 ± 0.5 control) and post-treatment (1.8 ± 0.7 vs. 6.8 ± 0.6 control) co...

European Journal of Cancer, 2014