Zulqarnain Mohamed

DNA-based characterisation and classification of forensically important flesh flies (Diptera: Sarcophagidae) in Malaysia.

Insect larvae and adult insects found on human corpses provide important clues for the estimation of the postmortem interval (PMI). Among all necrophagous insects, flesh flies (Diptera: Sarcophagidae) are considered as carrion flies of forensic importance. DNA variations of 17 Malaysian, two Indonesian and one Japanese flesh fly species are analysed using the mitochondrial COI and COII. These two DNA regions were useful for identifying most species experimented. However, characterisation of the species was not sufficiently made in the case of Sarcophaga javanica. Seventeen Malaysian species of forensic importance were successfully clustered into distinct clades and grouped into the six species groups: peregrina, albiceps, dux, pattoni, princeps and ruficornis. These groups correspond with generic or subgeneric taxa of the subfamily Sarcophaginae: Boettcherisca, Parasarcophaga, Liosarcophaga, Sarcorohdendorfia-Lioproctia, Harpagophalla-Seniorwhitea and Liopygia. The genetic variations found in COI and COII can be applied not only to identify the species of forensic importance, but also to understand the taxonomic positions, generic or subgeneric status, of the sarcophagine species.

Occurrence of Oriental Flies Associated with Indoor and Outdoor Human Remains in the Tropical Climate of North Malaysia

ABSTRACT: Flies attracted to human remains during death investigations were surveyed in north Peninsular Malaysia. Six families, eight genera, and 16 species were identified from human remains, with the greatest fly diversity occurring on remains recovered indoors. The total relative frequency of species was led by Chrysomya megacephala (Fabricius, 1794) (46%), followed by Chrysomya rufifacies (Macquart, 1842) (22%), Sarcophaga (Liopygia) ruficornis (Fabricius, 1974) (5%), Sarcophaga spp. (4%), Synthesiomyia nudiseta Wulp, 1883 (6%), Megaselia spp. (3%), Megaselia scalaris (Loew, 1866), (2%), Megaselia spiracularis Schmitz, 1938 (2%), and Chrysomya villeneuvi Patton, 1922 (2%). Hemipyrellia tagaliana (Bigot, 1877), Desmometopa sp., Megaselia curtineura (Brues, 1909), Hemipyrellia ligurriens Wiedemann 1830, Ophyra sp., Sarcophaga princeps Wiedemann 1830, Piophila casei (Linnaeus, 1758), and unidentified pupae each represented 1%, respectively.

A combination of doxycycline and ribavirin alleviated chikungunya infection

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.

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.

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.

In planta PCR-based detection of early infection of plant-parasitic nematodes in the roots: a step towards the understanding of infection and plant defence

The polyphagous obligate parasites Meloidogyne spp. devastate a wide range of crop plants including bananas and plantains. Their infestations impact agriculture worldwide. Therefore, an effective combating regime against this nematode species and an in-depth understanding of plant-nematode interaction are essential. Early detection of infection by visual inspection is not possible. This hampers early control strategy efforts and makes in-depth research of the early infection and plant defence unfeasible. A simple and robust in planta PCR-based nematode detection method is described here as the first crucial step. This PCR-based detection assay exploits the existence of the Internal Transcribed Spacer 1 (ITS 1) region of the ribosomal DNA (rDNA) gene family in the nematodes for early detection of nematode penetration into the roots. The results demonstrate that this detection assay is suitable to serve as a molecular screening tool for plant root diagnostic purposes.

A comparative study on the expression, purification and functional characterization of human adiponectin in Pichia pastoris and Escherichia coli.

Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.

Linkage of a medium sized Scottish autosomal dominant retinitis pigmentosa family to chromosome 7q.

Retinitis pigmentosa is a group of hereditary retinopathies which is both clinically and genetically heterogeneous. Autosomal dominant (ADRP), autosomal recessive (ARRP), and X linked recessive (XLRP), as well as digenic forms of inheritance have been reported. ADRP has been linked to 3q, 6p, 7p, 7q, 8cen, 17p, 17q, and 19q. Three unrelated ADRP families have been reported to show linkage to 7q. We tested a Scottish ADRP family with microsatellite markers mapping within the 7q31-q35 region, and found three markers (D7S487, D7S514, D7S530) showing statistically significant evidence of linkage. A maximum two point lod score of 3.311 at 0% recombination was obtained for D7S514.

Mefenamic acid in combination with ribavirin shows significant effects in reducing chikungunya virus infection in vitro and in vivo

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.

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.