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1.  Combination of VP3 and CD147-knockdown enhance apoptosis and tumor growth delay index in colorectal tumor allograft 
BMC Cancer  2016;16:461.
Cancer therapies that kill cancer cells without affecting normal cells is the ultimate mode of treating cancers. The VP3, an avian virus-derived protein, can specifically initiate cell death through several signal transduction pathways leading to apoptosis. In cancer, chemoresistance and cell survivability implicate the cell surface protein, CD147.
In this study, transfection of VP3 and silencing of CD147 genes was achieved through the treatment of tumors with pVIVO1-GFP/VP3 (VP3), psiRNA-CD147/2 (shCD147/2), and their combination of CT26 colon cancer cell-induced in mice. The effectiveness of tumor-treatment was ascertained by electrophoresis, TUNEL assay, and flow cytometry analysis. While histopathological and biochemical analysis were used as toxic side effect identification.
The tumor growth delay index (TGDI) after treatment with VP3, shCD147/2, and their combination treatments increased by 1.3-, 1.2-, 2.0- and 2.3-fold respectively, over untreated control. The VP3-shCD147/2 combination treatment was more efficacious then either VP3 or shCD147/2 alone in the retardation of mouse CT26 colorectal cell tumor allograft.
The antitumor effect of the combination treatment is the result of synergistic effects of VP3 and shCD147/2 on the tumor cells resulting in apoptosis. Thus, the study shows that combination of VP3 and shCD147/2 treatment can be developed into a potential approach for anticolorectal cancer treatment regimen.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-016-2530-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4944445  PMID: 27411985
pVIVO1-GFP/VP3; psiRNA-CD147/2; CT26 colon cancer cell tumor; Apoptosis
2.  Antiviral and virucidal activities of Duabanga grandiflora leaf extract against Pseudorabies virus in vitro 
Duabanga grandiflora or known in Malaysia as Berembang Bukit, Megawasih, or Pedada Bukit, is a native plant of the Southeast Asian countries. In this study, the anti-viral properties of D. grandiflora were investigated.
The D. grandiflora leaf extracts were obtained with ethyl acetate, hexane, and ethanol as solvents and labelled 37 leaf ethyl acetate (37 L EA), 37 leaf hexane (37 L H), 37 leaf ethanol (37 L ET), respectively. The cytotoxicity of the extracts on Vero cells were determined by the 3-(4,5-Diamethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.
Among extracts, 37 L EA was most cytotoxic to Vero cells, followed by 37 L H and 37 L ET, with CC50 of 218, 833, and >1000 μg/mL, respectively. The cytopathic effect (CPE) and plaque reduction, inhibition, and virucidal assays and the selective index (SI) were employed to determine the effect of the extracts on infectivity and replication of pseudorabies virus (PrV) in Vero cells. The D. grandiflora leaf extracts showed dose-dependent antiviral activities, with higher activities at high doses. The 37 L ET and 37 L EA showed anti-viral effects through plaque formation and viral replication inhibitions, and virucidal property. The SI of the 37 L ET and 37 L EA by the viral replication inhibition assay was 8.3 and 1.9, respectively, and by the CPE reduction assay, 6.7 and 2.9, respectively.
Ethanol is the best solvent for the preparation of D. grandiflora leaf extract as an antiviral agent.
Electronic supplementary material
The online version of this article (doi:10.1186/s12906-016-1120-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4877979  PMID: 27216794
Antiviral assay; Duabanga grandiflora; Plaque reduction assay; Inhibition assay; Virucidal assay
3.  Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin 
BMC Cancer  2013;13:488.
Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. Five truncated Apoptin proteins were analyzed to determine their selective ability to migrate into the nucleus of human breast adenocarcinoma MCF-7 cells for inducing apoptosis.
For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N’ terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect.
Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32–83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1–31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin’s signature targeting activity.
Therefore, the critical stretch spanning amino acid 1–31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.
PMCID: PMC4015422  PMID: 24144306
VP3; Apoptin; MCF7 cells; Chang cells; Apoptosis; Microinjection; Truncation
4.  Phylogenetic characterization of genes encoding for viral envelope glycoprotein (ORF5) and nucleocapsid protein (ORF7) of porcine reproductive & respiratory syndrome virus found in Malaysia in 2013 and 2014. 
Porcine reproductive and respiratory syndrome (PRRS) is one of the most expensive diseases of modern swine production & results in annual economic losses and cost the industry over 600 million USD in U.S. alone and billions of dollars worldwide. Two atypical PRRS cases were observed in 2013 and 2014 characterized by late-term abortion, fever and sudden increase in sow mortality which persisted for a prolonged period of time.
Lungs, lymph nodes and other samples were collected for disease investigation. Sequencing of the viral envelope glycoprotein (ORF5) and nucleocapsid protein (ORF7) of PRRSV was done using the BigDye Terminator v3.1 cycle sequencing kit chemistry. The phylogenetic tree was constructed by using the Maximum Likelihood method, generated by Mega 6.06®.
Analysis of the ORF5 and ORF7 showed high degree of sequence homology to PRRSV parent vaccine strain VR-2332, RespPRRSV and other mutant/chimeric virus strains.
Our study suggests that recombination events between vaccine strains and field isolates may contribute to PRRSV virulence in the field.
PMCID: PMC5217455  PMID: 28056965
Porcine reproductive and respiratory syndrome virus; PRRSV; Genetic characterization; ORF5 gene; ORF7 gene
5.  Observation of risk factors, clinical manifestations and genetic characterization of recent Newcastle Disease Virus outbreak in West Malaysia 
BMC Veterinary Research  2015;11:219.
Newcastle disease virus remains a constant threat in commercial poultry farms despite intensive vaccination programs. Outbreaks attributed to ND can escalate and spread across farms and states contributing to major economic loss in poultry farms.
Phylogenetic analysis in our study showed that eleven of the samples belonged to genotype VIId. All farms were concurrently positive with two immunosuppressive viruses; Infectious Bursal Disease Virus (IBDV) and Marek’s Disease Virus (MDV). Amino acid sequence analysis confirmed that eleven of the samples had sequence motifs for velogenic/mesogenic strains; three were lentogenic.
In conclusion, no new NDV genotype was isolated from the 2011 NDV outbreak. This study suggests that the presence of other immunosuppressive agents such as IBD and MDV could have contributed to the dysfunction of the immune system of the chickens, causing severe NDV outbreaks in 2011. Risk factors related to biosecurity and farm practices appear to have a significant role in the severity of the disease observed in affected farms.
PMCID: PMC4546084  PMID: 26293577
Newcastle disease virus; Infectious Bursal Disease; Marek’s Disease; Immunosuppressive agents; Recent outbreak; Risk factors; Phylogenetic study; Genetic characterization
6.  Reduction of MTT to Purple Formazan by Vitamin E Isomers in the Absence of Cells 
Tropical Life Sciences Research  2015;26(1):111-120.
The yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is widely used to determine cell viability in cell proliferation and cytotoxic assays. MTT is reduced by metabolically active cells to form an insoluble purple formazan product that is quantifiable by spectrophotometry. It is the most common and direct assay for cell viability. However, in this present study, we demonstrated that the vitamin E isomers α-β-γ-δ-tocotrienols and α-tocopherol were able to reduce MTT into a formazan product, despite the absence of living cells. For comparison, a second method for determining cell viability, which is the neutral red uptake assay, was used in parallel with the MTT assay. The results showed that neutral red did not interact with the vitamin E isomers. Our findings suggest that the MTT assay is not suitable for studying the proliferative effects of vitamin E isomers on cell growth.
PMCID: PMC4437321
MTT; Neutral Red Uptake; Vitamin E Isomers; Cell Viability; MTT; Penyerapan Neutral Merah; Isomer-isomer Vitamin E; Daya Maju Sel
7.  Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology 
BMC Biotechnology  2012;12:70.
Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.
The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.
The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.
PMCID: PMC3487952  PMID: 23039947
Hepatitis B surface antigen; Cell disruption; Glass bead; High pressure homogenizer; Pichia pastoris; Recombinant protein

Results 1-7 (7)