Development of a Deoxyribonucleic Acid Vaccine Against Enterovirus 71

Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand,foot and mouth disease (HFMD), a common rash illness in children and infants.There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and constructa DNA vaccine against Enterovirus 71 using the viral capsid protein(VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo.The VP1 gene of EV71 isolate S2/86/1 and isolate 410/4 obtained from Prof.Mary Jane Cardosa,Universiti Malaysia Sarawak (UNIMAS) were amplified using PCR and then inserted into a eukaryotic expression vector,pVAX1.The 3.9 kb recombinant constructs were transformed into competent E.coli cells and the positive clones were screened and selected using PCR analysis,restriction digestion analysis and DNA sequencing. The pVAX1 vector that was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame, were designated as pVAX1/VP1-S and pVAX1/VP1-4,respectively.The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The constructs were then tested for protein expression in Vero cells.The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting.Subsequently,in the in vivo studies,female Balb/c mice were immunized with the DNA vaccine constructs.Enzyme Linked Immunosorbent Assay (ELISA) was performed to detect the presence of anti-VP1 IgG in mice.The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster.The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71.The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccin

Toll-like receptor 9 and 4 gene polymorphisms in susceptibility and severity of malaria: a meta-analysis of genetic association studies

Background: Malaria is still a major public health problem in sub-Saharan Africa and South-east Asia. The clinical presentations of malaria infection vary from a mild febrile illness to life-threatening severe malaria. Toll like receptors (TLRs) are postulated to be involved in the innate immune responses to malaria. Individual studies showed inconclusive findings. This study aimed to assess the role of TLR4 (D299G, T399I) and TLR9 (T1237C, T1486C) in severity or susceptibility of malaria by meta-analysis of data from eligible studies. Methods: Relevant case–control studies that assessed the association between TLR 4/9 and malaria either in susceptibility or progression were searched in health-related electronic databases. Quality of included studies was evaluated with Newcastle–Ottawa scale. Pooled analyses for specific genetic polymorphisms were done under five genetic models. Stratified analysis was done by age and geographical region (Asian countries vs non-Asian countries). Results: Eleven studies (2716 cases and 2376 controls) from nine endemic countries were identified. Five studies (45.4%) obtained high score in quality assessment. Overall, a significant association between TLR9 (T1486C) and severity of malaria is observed in allele model (OR: 1.26, 95% CI: 1.08–1.48, I2 = 0%) or homozygous model (OR: 1.55, 95% CI: 1.08–2.28, I2 = 0%). For TLR9 (T1237C), a significant association with severity of malaria is observed in in heterozygous model (OR:1.89, 95% CI: 1.11–3.22, I2 = 75%). On stratifications, TLR9 (T1486C) is only significantly associated with a subgroup of children of non-Asian countries under allele model (OR: 1.25, 95% CI: 1.02–1.38), while 1237 is with a subgroup of adults from Asian countries under heterozygous model (OR: 2.0, 95% CI: 1.09–3.64, I2 = 39%). Regarding the susceptibility to malaria, TLR9 (T1237C) is significantly associated only with the children group under recessive model (OR: 2.21, 95% CI: 1.06–4.57, I2=85%) and homozygous model (OR: 1.49, 95% CI: 1.09–2.0, I2 = 0%). For TLR4 (D299G, T399I), none is significantly associated with either severity of malaria or susceptibility to malaria under any genetic models. Conclusions: The findings suggest that TLR 9 (T1486C and T1237C) seems to influence the progression of malaria, under certain genetic models and in specific age group of people from specific geographical region. TLR 9 (T1237C) also plays a role in susceptibility to malaria under certain genetic models and only with children of non-Asian countries. To substantiate these, future well designed studies with larger samples across endemic countries are needed

An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads

This report describes a modified, cost-effective method of cell wall disruption for the yeast Candida spp., which employs the use of glass beads in a simple sorbitol lysis buffer. This method can be used in conjunction with a commercial RNA or genomic DNA isolation method to obtain high-quality RNA or DNA

Antioxidant and Toxicity Studies of 50% Methanolic Extract of Orthosiphon stamineus Benth

The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between bothmale and female treated rats in any doses tested.No abnormality of internal organs was observed between treatment and control groups.Theoral lethal dose determined wasmore than 5000mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000mg/kg per day

Antioxidant and toxicity studies of 50% methanolic extract of Orthosiphon stamineus benth.

The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000 mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000 mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between both male and female treated rats in any doses tested. No abnormality of internal organs was observed between treatment and control groups. The oral lethal dose determined was more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000 mg/kg per day

Morphology and growth of carbon nanotubes catalytically synthesised by premixed hydrocarbon-rich flames

Synthesis of carbon nanotubes (CNTs) was performed by using a laminar premixed flame burner at open atmospheric condition. The growth of CNTs on the substrate was supported catalytically by a transition metal under high temperature, hydrocarbon-rich environment. Analysis of the CNTs using high resolution electron microscope reveals the structure of synthesised nano-materials in disarray, clustered and tubular form. The graphitic structure of the CNTs are rather similar for all fuel-rich equivalence ratios tested, with an average diameter of ∼11–13 nm. Removal of the amorphous carbon and catalyst in the CNTs was performed via purification treatment using H2O2 and HCl solutions. Detail characterisation indicates the oxidation temperature of purified CNTs ranges between 497 and 529 °C. Deconvolution of the Raman spectra in the range of 900–1800 cm−1 shows the distinct characteristic bands of CNTs with IG/ID ratio of 0.66–0.72 for all the samples tested. In addition, the high level carbon concentration and sp2 Csingle bondC bond in the CNTs is shown by X-ray photoelectron spectroscopy analysis. The present study demonstrates that CNTs can be effectively synthesised from fuel-rich hydrocarbon flames at ϕ = 1.8–2.0 supported by nickel-based substrate

Insulin resistance, inflammation and metabolic syndrome in normal weight and overweight/obese primary school children in Kuala Lumpur

Introduction: Studies on metabolic syndrome (MetS) of children are important in view of rising prevalence of childhood obesity worldwide. This study compares the risks of insulin resistance, inflammation and metabolic syndrome between overweight/obese (OW/OB) and normal weight (NW) children in Kuala Lumpur. Methods: A crosssectional study was conducted in 12 primary schools selected using multi-stage stratified random sampling. Height and weight were taken of a total of 1971 children aged 10-11 years. Based on BMI-for-age, 235 OW/OB children matched for age, sex and ethnicity with 226 NW children were selected for the study. Overnight fasting blood samples were collected to determine insulin, high-sensitivity C-reactive protein (hsCRP), glucose and lipid profiles. Logistic regression analysis was conducted to estimate associations between weight status and metabolic risk factors. Results: Prevalence of MetS among OW/OB children was 3.8% compared to 0% in the NW. Prevalence of insulin resistance among OW/OB was 45.5% compared to 18.6% among NW children. High risk of inflammation was found in 28.1% of the OW/OB children compared to 12.4% in the NW. The odds ratio of having insulin resistance, inflammation and metabolic risk factors among OW/OB were 3.66 (95% CI: 2.40-5.59), 2.76 (95% CI: 1.69-4.50), 4.93 (95% CI: 3.42-7.10), respectively compared to the NW. Conclusion: The OW/OB children in this study showed higher risks of developing insulin resistance, inflammation and MetS compared to the NW counterparts. Further studies are suggested to better understand the relationships between insulin resistance, inflammation and MetS in children

Marine micro-phytoplankton of Singapore, with a review of harmful microalgae in the region

A survey of marine phytoplankton in the Singapore Strait was carried out between May and June 2013, as part of an effort to determine the diversity of phytoplankton in Singapore’s coastal waters. A total of 34 microalgal samples were collected using a 20 μm-mesh plankton net and from coastal sediments. Living samples and preserved samples in Lugol’s solution were identified to species as far as possible under the microscope. A checklist of marine micro-phytoplankton was updated to encompass 270 taxa, including 49 new records from Singapore waters. Some 37 species from 15 families were dinoflagellates, and 233 species from 50 families were diatoms. Harmful microalgae, categorized as biotoxin-producers and fish killers, were also found in this survey. These were in the genera Alexandrium, Amphidinium, Ceratium, Cochlodinium, Coolia, Dinophysis, Gambierdiscus, Karenia, Karlodinium, Ostreopsis, Prorocentrum, Nitzschia, and Pseudo-nitzschia