Partial Purification and Characterisation of Alkaline Phosphatase from Hepatopancreas and Intertine of Red Tilapia, (Tilapia Mossambica)

Alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyze nonspecific hydrolysis of phosphate monoesters. Partial purification was conducted on alkaline phosphatase (ALP) extracted from hepatopancreas and intestine of red tilapia, (Tilapia mossombica) using two main steps - ammonium sulphate precipitation and ion exchange chromatography on DEAE - 52. Samples from the ion-exchange step were analysed for ALP activities and characterised by SDS-PAGE. SDS-PAGE analysis showed 2 identical bands and was found to have molecular weight of 68,000 Da (hepatopancreas ALP) and 180,500 Da (intestinal ALP) subunits. Overall, purification fold obtained from the final step are 1.8 and 21.9 for hepatopancreas and intestinal respectively, with recovery of only 0.22% from hepatopancreas and 0.01% from intestine. The specific activity of the enzyme was 1.72 X 10- 2 pmol min-1 mg-1 and 2.93 X 10-1 pmol min-1 mg-1 from hepatopancreas and intestine respectively. The ALP from hepatopancreas remained stable at temperatures up to 50°C, and ALP from intestine enzyme had an optimum temperature of 60°C. The optimum pH for both hepatopancreas and intestine ALP of Tilapia mossambica is pH 10. The positive monovalent alkali metal ions (Li+, Na+ and K+) have no effect on the ALP enzyme activity. However, the positive divalent alkali metal ions (Mg2+a nd Ca2+)a ctivate the enzyme activities. Heavy metal ions (Zn2+, CU~+C,d 2+a nd Hg2+) were found to inhibit the enzyme activity

Profiling of Burkholderia cepacia Secretome at Mid-Logarithmic and Early-Stationary Phases of Growth

BACKGROUND: Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence. CONCLUSION/SIGNIFICANCE: Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release

Step by step preparation of urine samples for in-Solution, gel-free proteomic studies, suitable for MALDI TOF MS and LCMS QTOF MS

Two-dimensional electrophoresis is an established method for isolating and separating proteins for proteomic analysis and identification of proteins. However, it requires a lot of optimization and it suffers from some ongoing concerns regarding quantitative reproducibility and limitations on its ability to study certain class of proteins. We propose a simpler method in preparing urine samples from acute melioidosis patients for in-solution proteomics using liquid chromatography coupled with mass spectrometry quadrupole time of flight (LCMS-QTOF MS) or matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This method is relatively easier than 2-dimensional electrophoresis, affordable, reproducible, can be performed in less equipped laboratories for transport to specialized centers for proteomic studies and bioinformatic analysis

Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B)

Genotypic and phenotypic characterization of Escherichia coli isolated from indigenous individuals in Malaysia

Objective(s): The occurrence of asymptomatic verocytotoxin (VT)-producing Escherichia coli (VTEC) infections among humans in recent years is posing a high risk to public health. Thus, the role of asymptomatic human carriers as a source of dissemination should not be underestimated. This study aimed to elucidate the phenotypic and genotypic characteristics of E. coli in the stool samples collected from indigenous individuals in Malaysia. Materials and Methods: E. coli strains (n=108) were isolated from stool samples obtained from 41 indigenous individuals. All strains were subjected to Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) typing and confirmation of VTEC variants. Non-duplicate strains were selected based on REP-PCR profiles and further subjected to antimicrobial susceptibility test (AST). The genotypic and phenotypic characteristics of the strains were then correlated with the demographic data of the subjects. Results: A total of 66 REP-PCR profiles grouped in 53 clusters (F=85%) were obtained. Four genetically distinct strains were confirmed as VTEC (eaeA-positive). The predominant resistance was against ampicillin (34.2%), followed by trimethoprim-sulfamethoxazole (32.9%), ampicillin-sulbactam (5.5%), and ciprofloxacin (1.4%). All isolates were sensitive to amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, and meropenem. Conclusion: Genetically diverse E. coli and VTEC strains were found to colonize the intestines of the indigenous populations. This study is important for the prospective surveillance of E. coli among the indigenous individuals in Malaysia, especially in asymptomatic VTEC infection and antimicrobial resistance phenomenon

Association between vision and cognitive function among community-dwelling older adults in Selangor, Malaysia

AIM: To determine the association between distance and near visual acuity (VA) and cognitive function among older adults in Selangor, Malaysia. METHODS: A total of 230 older adults (age ≥60y) participated in this study. Habitual distance and near VA were measured using the Early Treatment Diabetic Retinopathy Study Chart and Lighthouse Near Visual Acuity Chart, respectively. Global cognitive function was assessed using the Mini-Mental State Examination (M-MSE) and the Malay language version of the Montreal Cognitive Assessment (M-MoCA). Digit Symbol (DS) subtest was used to measure information processing. RESULTS: No significant association was observed between vision and M-MSE and M-MoCA scores. However, poor distance and near VA were found to be significantly associated with low DS scores [distance VA: β=-0.01, R2=0.1, P=0.02; odds ratio (OR)=2.84, 95% confidence interval (CI), 1.10-7.33, P=0.03; near VA: β=-0.05, R2=0.08, P=0.00; OR=3.32, 95%CI, 1.28-8.59, P=0.01]. CONCLUSION: Poor vision is associated with a decline in information processing in older adults and substantiates the importance of preserving good vision in maintaining cognitive function

Association between knowledge, attitude, and practice of nutrition and food labels among selected higher educational institution students in Klang Valley

Nutrition information on food labels guides consumers to purchase healthier food choices. Besides nutrition information, other factors influence a purchase. This study aims to determine the association between the knowledge, attitude, and practice (KAP) among tertiary students on nutrition and food labels. In this cross-sectional study, a total of 190 students from three tertiary institutions within Klang Valley completed an online survey. Self-administered questionnaires on sociodemographic profiles and KAP questions, available in Malay and English, were distributed. Association between KAP was determined using Spearman's Rho test, while multiple linear regression was used to assess predictors of KAP scores. Mean body mass index (BMI) of the respondents were 20.8 kg/m2. The total mean score for knowledge on food labels was 8.93, followed by attitude and practice with 3.86 and 3.11, respectively. There was a significant correlation between attitude and practice (p<0.005). Nutrient and total calorie information on food labels influenced purchases, with 56.3% of respondents reported looking at the total calorie content, followed by 55.7% and 49.5% checking on sugar and fats, respectively. In addition, other factors such as expiry date (60.9%) and price (59.9%) also influenced purchases. Overall, respondents have a positive attitude on food selection, but male respondents have better knowledge levels than females. However, female respondents interpret food labelling effectively compared to male respondents. Despite having good knowledge and attitude towards nutrition, respondents were still making poor choices. A more extensive range of healthier food options and targeted healthy eating campaigns may empower students to choose more nutritious foods

Burkholderia pseudomallei short-chain dehydrogenase/ oxidoreductase: potential urine biomarker candidate for acute melioidosis

INTRODUCTION: In the current climate of urgency in identifying biomarkers for the development of rapid diagnostic kits, the use of urine samples to diagnose acute melioidosis was evaluated, comparing urine samples from Burkholderia pseudomallei culture-positive and culture-negative patients, and comparing pneumonic and septicemic melioidosis. MATERIAL AND METHODS: Eleven urine samples from clinically suspected melioidosis patients from a tertiary referral center, Hospital Tengku Ampuan Afzan, Pahang was used. An in-solution method for the detection of bacterial proteins using liquid chromatography-mass spectrometry quadrupole time-of-flight (LCMS QTOF) was used. RESULTS: Three bacterial proteins were consistently detected among all the culture[1]positive and PCR-positive cases tested, namely SDR family NAD(P)-dependent oxidoreductase protein (32kDa), 3-hydroxyacyl-CoA dehydrogenase Burkholderia sp. (32kDa), and NAD(P)-dependent dehydrogenase (short-subunit alcohol dehydrogenase family) Burkholderia sp. (33kDa). CONCLUSIONS: Short-chain dehydrogenase (SDO) proteins could potentially be a urine biomarker candidate as these have shown to aid in the ability of Burkholderia spp. to invade host cells as this action is important for the initial intracellular survival of the organism

Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells.

BACKGROUND:Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. METHODS:We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). RESULTS:We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. CONCLUSION:Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections

Optimized preparation of urine samples from acute melioidosis patients for in-solution proteomic studies using LCMS QTOF or MALDI TOF MS

Introduction: Investigation of urine proteome in patients with acute melioidosis may reveal potential disease markers, from either bacterial or human proteins. We used an in-solution gel-free method instead of 2-DE to detect human and Burkholderia pseudomallei proteins in urine of patients with acute melioidosis. Here, we propose a simpler, economical method for preparing urine samples directly from melioidosis patients, for in-solution proteomic analysis using LCMS-QTOF MS/MS or MALDI-TOF MS/MS. Material and Methods: We adapted an acetone-TCA based protein precipitation method with LCMS-QTOF MS to detect the B. pseudomallei proteins directly from urine of acute melioidosis patients (culture positive and negative). This process involves protein precipitation, desalting, trypsin digestion, and optimization for the mass spectrometry. Results: A total of 3,866 human peptides were detected across 11 urine samples from clinically suspected acute melioidosis patients. Among them were three Burkholderia specific proteins detected in 75% of culture positive samples. Large amounts of acute phase proteins, cell mediated immunity proteins, complement pathway proteins and inflammatory mediators were seen upon gene ontology (GO) annotation and GO enrichment analysis. Conclusions: This simple in-solution sample preparation method can be replicated easily for LCMS/MS-QTOF and MALDI-TOF proteomic analyses, avoiding tedious optimization steps in 2-DE. This method is cost effective and can be done in centres without specialized 2-DE or MS equipment and elutes can be easily transported for analysis and bioinformatics. This is the first study to analyse urine samples directly for B. pseudomallei proteins. Discovery of the entire proteome as a whole is important in leading to biomarker discovery