Examination of Cold-Plaque Screening Technique as a Means to Isolate Low Abundance Genes from Oil Palm (Elaeis Guineensis) Flowers

Genes that are present at low abundance in cells are difficult to clone by using standard molecular biology techniques such as conventional differential screening. In plants, many of these low abundance genes encode transcription factors or proteins involved in signal tranduction. Therefore in this study, a cold-plaque screening technique was used as a means to enrich for low abundance genes from an oil palm male flower cDNA library. When a total of 441 non-hybridising plaques ('cold' plaques) were isolated, 123 clones (opcp population) were found to contain inserts with a minimal size of 500 basepairs and were independent clones. Initial screening of these clones by reverse northern analysis with the same probes used during differential screening and an additional probe from female flower of 6 em showed that these 0PCP clones could be categorised into five subpopulations based on their tissue-specificity and expression levels. 61.8% of the 123 clones were expressed at high abundance with all the three probes (Subpopulation A)whilst 4.1% of the clones were lowly-expressed in both male and female flower tissues (Subpopulation B). 7.3% of the clones were expressed at medium abundance but were male-predominant (Subpopulation C) while 11.4% of the clones were expressed at low abundance and were male-predominant (Subpopulation D) and 15.4% of the clones did not show any detectable expression with any of the probes used (Subpopulation E). Partial sequencing of all clones from subpopulation B, C, D and E as well as eight clones from subpopulation A showed that opcp72 (subpopulation D) is a putative UIP2 (Unusual Floral Organ (UFO) binding protein) homolog, opcpl44 (subpopulation A) encodes elongation factor-la, opcp327 (subpopulation E) encodes a putative RLK 5 (Receptor-like Protein Kinase) homolog and opcp441 (subpopulation A) is a putative fructose 1, 6-bisphosphate aldolase gene. Expression studies on ten opcp clones with two representative clones from each subpopulation showed similar expression profiles where hybridisation signals were detected at the early flower development with higher signals in the meristem tissues but no detectable hybridisation signals in 3.5 em and 6 em male flower, one of the stages used to make the male flower cDNA library. Southern hybridisation of genomic DNA for clone opcp72, opcpl44 and opcp327 showed that these genes are low copy genes.In conclusion the use of cold-plaque screening techniques can result in the isolation of a variety of clones whose expression ranges from low abundance (undetectable in the Northern blots), to those that are lowly expressed during the stages of floral development used to construct the oil palm male-flower cDNA library

Does use of pooled cohort risk score overestimate the use of statin?: a retrospective cohort study in a primary care setting

BACKGROUND: Initiation of statin therapy as primary prevention particularly in those with mildly elevated cardiovascular disease risk factors is still being debated. The 2013 ACC/AHA blood cholesterol guideline recommends initiation of statin by estimating the 10-year atherosclerotic cardiovascular disease (ASCVD) risk using the new pooled cohort risk score. This paper examines the use of the pooled cohort risk score and compares it to actual use of statins in daily clinical practice in a primary care setting. METHODS: We examined the use of statins in a randomly selected sample of patients in a primary care clinic. The demographic data and cardiovascular risk parameters were captured from patient records in 1998. The pooled cohort risk score was calculated based on the parameters in 1998. The use of statins in 1998 and 2007, a 10-year interval, was recorded. RESULTS: A total of 847 patients were entered into the analysis. Mean age of the patients was 57.2 ± 8.4 years and 33.1% were male. The use of statins in 1998 was only 10.2% (n = 86) as compared to 67.5% (n = 572) in 2007. For patients with LDL 70-189 mg/dl and estimated 10-year ASCVD risk ≥7.5% (n = 190), 60% (n = 114) of patients were on statin therapy by 2007. There were 124 patients in whom statin therapy was not recommended according to ACC/AHA guideline but were actually receiving statin therapy. CONCLUSIONS: An extra 40% of patients need to be treated with statin if the 2013 ACC/AHA blood cholesterol guideline is used. However the absolute number of patients who needed to be treated based on the ACC/AHA guideline is lower than the number of patients actually receiving it in a daily clinical practice. The pooled cohort risk score does not increase the absolute number of patients who are actually treated with statins. However these findings and the use of the pooled cohort risk score need to be validated further

Visit-to-visit SBP variability and cardiovascular disease in amultiethnic primary care setting:10-year retrospective cohort study

Objectives: The current study aims to determine the relationship of long-term visit-to-visit variability of SBP to cardiovascular disease (CVD) in a multiethnic primary care setting. Method: This is a retrospective study of a cohort of 807 hypertensive patients over a period of 10 years. Three-monthly clinic blood pressure readings were used to derive blood pressure variability (BPV), and CVD events were captured from patient records. Results: Mean age at baseline was 57.2 ± 9.8 years with 63.3% being women. The BPV and mean SBP over 10 years were 14.7 ± 3.5 and 142 ± 8 mmHg, respectively. Prevalence of cardiovascular event was 13%. In multivariate logistic regression analysis, BPV was the predictor of CVD events, whereas the mean SBP was not independently associated with cardiovascular events in this population. Those with lower SBP and lower BPV had fewer cardiovascular events than those with the same low mean SBP but higher BPV (10.5 versus 12.8%). Similarly those with higher mean SBP but lower BPV also had fewer cardiovascular events than those with the same high mean and higher BPV (11.6 versus 16.7%). Other variables like being men, diabetes and Indian compared with Chinese are more likely to be associated with cardiovascular events. Conclusion: BPV is associated with an increase in CVD events even in those who have achieved lower mean SBP. Thus, we should prioritize not only control of SBP levels but also BPV to reduce CVD events further

Number of blood pressure measurements needed to estimate long-term visit-to-visit systolic blood pressure variability for predicting cardiovascular risk: a 10-year retrospective cohort study in a primary care clinic in Malaysia

Objective: To determine the reproducibility of visit-to-visit blood pressure variability (BPV) in clinical practice. We also determined the minimum number of blood pressure (BP) measurements needed to estimate long-term visit-to-visit BPV for predicting 10-year cardiovascular (CV) risk. Design: Retrospective study Setting: A primary care clinic in a university hospital in Malaysia. Participants: Random sampling of 1403 patients aged 30 years and above without any CV event at baseline. Outcomes measures: The effect of the number of BP measurement for calculation of long-term visit-to-visit BPV in predicting 10-year CV risk. CV events were defined as fatal and non-fatal coronary heart disease, fatal and non-fatal stroke, heart failure and peripheral vascular disease. Results: The mean 10-year SD of systolic blood pressure (SBP) for this cohort was 13.8±3.5 mm Hg. The intraclass correlation coefficient (ICC) for the SD of SBP based on the first eight and second eight measurements was 0.38 (p<0.001). In a primary care setting, visit-to-visit BPV (SD of SBP calculated from 20 BP measurements) was significantly associated with CV events (adjusted OR 1.07, 95% CI 1.02 to 1.13, p=0.009). Using SD of SBP from 20 measurement as reference, SD of SBP from 6 measurements (median time 1.75 years) has high reliability (ICC 0.74, p<0.001), with a mean difference of 0.6 mm Hg. Hence, a minimum of six BP measurements is needed for reliably estimating intraindividual BPV for CV outcome prediction. Conclusion: Long-term visit-to-visit BPV is reproducible in clinical practice. We suggest a minimum of six BP measurements for calculation of intraindividual visit-to-visit BPV. The number and duration of BP readings to derive BPV should be taken into consideration in predicting long-term CV risk

Development of high resolution melting analysis for the diagnosis of human malaria

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis

Medication adherence, its associated factors and implication on glycaemic control in patients with type 2 diabetes mellitus: A cross-sectional study in a Malaysian primary care clinic

Introduction: Medication adherence and metabolic control remain suboptimal among patients with diabetes mellitus in Malaysia despite the clear benefits of reduced vascular complications and mortality risk. This study examined the factors associated with medication adherence and glycaemic control in patients with type 2 diabetes mellitus in a primary care clinic. Methods: This cross-sectional study was conducted in a public health clinic in Pagoh, Johor, among 386 patients recruited via systematic random sampling. Data were obtained using a validated 7-item structured questionnaire, glycated haemoglobin (HbA1c) test and medical record review. Logistic regression analysis was performed to determine the factors associated with medication adherence. Results: The mean patient age was 60.04±10.75 years, and the mean HbA1c level was 8.3±2.0%. Approximately 60.3% of the participants were adherent to their medication, and an increasing age was significantly associated with medication nonadherence (adjusted odds ratio [OR]: 0.959; confidence interval [CI]: 0.934–0.985). Medication adherence (adjusted OR: 2.688; CI: 1.534–4.708) and use of combined oral medications (adjusted OR: 5.604; CI: 3.078–10.203), combined oral medications with insulin (adjusted OR: 23.466; CI: 8.208–67.085) and insulin only (adjusted OR: 6.528; CI: 1.876–22.717) were associated with good glycaemic control. Older age (adjusted OR: 0.954; CI: 0.923–0.986) and Malay ethnicity (adjusted OR: 0.284; CI: 0.101–0.794) were associated with poor glycaemic control. Conclusion: Suboptimal medication adherence and glycaemic control are prevalent in primary care settings, especially among elderly patients. Counselling should be targeted to patients and their caretakers to improve medication adherence and optimise metabolic control

Validation of the framingham general cardiovascular risk score in a multi-ethnic Asian population a retrospective cohort study

Objective This study aims to examine the validity of the Framingham general cardiovascular disease (CVD) risk chart in a primary care setting. Design This is a 10-year retrospective cohort study. Setting A primary care clinic in a teaching hospital in Malaysia. Participants 967 patients’ records were randomly selected from patients who were attending follow-up in the clinic. Main outcome measures Baseline demographic data, history of diabetes and smoking, blood pressure (BP), and serum lipids were captured from patient records in 1998. Each patient's Framingham CVD score was computed from these parameters. All atherosclerotic CVD events occurring between 1998 and 2007 were counted. Results In 1998, mean age was 57 years with 33.8% men, 6.1% smokers, 43.3% diabetics and 59.7% hypertensive. Median BP was 140/80 mm Hg and total cholesterol 6.0 mmol/L (1.3). The predicted median Framingham general CVD risk score for the study population was 21.5% (IQR 1.2–30.0) while the actual CVD events that occurred in the 10 years was 13.1% (127/967). The median CVD points for men was 30.0, giving them a CVD risk of more than 30%; for women it is 18.5, a CVD risk of 21.5%. Our study found that the Framingham general CVD risk score to have moderate discrimination with an area under the receiver operating characteristic curve (AUC) of 0.63. It also discriminates well for Malay (AUC 0.65, p=0.01), Chinese (AUC 0.60, p=0.03), and Indians (AUC 0.65, p=0.001). There was good calibration with Hosmer-Lemeshow test χ2=3.25, p=0.78. Conclusions Taking into account that this cohort of patients were already on treatment, the Framingham General CVD Risk Prediction Score predicts fairly accurately for men and overestimates somewhat for women. In the absence of local risk prediction charts, the Framingham general CVD risk prediction chart is a reasonable alternative for use in a multiethnic group in a primary care setting

Molecular detection of human Plasmodium species in Sabah using PlasmoNextm multiplex PCR and hydrolysis probes real-time PCR

BACKGROUND: Malaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Five Plasmodium species have been recognized by World Health Organization (WHO) as the causative agents of human malaria. Generally, microscopic examination is the gold standard for routine malaria diagnosis. However, molecular PCR assays in many cases have shown improvement on the sensitivity and specificity over microscopic or other immunochromatographic assays. METHODS: The present study attempts to screen 207 suspected malaria samples from patients seeking treatment in clinics around Sabah state, Malaysia, using two panels of multiplex PCRs, conventional PCR system (PlasmoNex™) and real-time PCR based on hydrolysis probe technology. Discordance results between two PCR assays were further confirmed by sequencing using 18S ssu rRNA species-specific primers. RESULTS: Of the 207 malaria samples, Plasmodium knowlesi (73.4% vs 72.0%) was the most prevalent species based on two PCR assays, followed by Plasmodium falciparum (15.9% vs 17.9%), and Plasmodium vivax (9.7% vs 7.7%), respectively. Neither Plasmodium malariae nor Plasmodium ovale was detected in this study. Nine discrepant species identification based on both the PCR assays were further confirmed through DNA sequencing. Species-specific real-time PCR only accurately diagnosed 198 of 207 (95.7%) malaria samples up to species level in contrast to PlasmoNex™ assay which had 100% sensitivity and specificity based on sequencing results. CONCLUSIONS: Multiplex PCR accelerate the speed in the diagnosis of malaria. The PlasmoNex™ PCR assay seems to be more accurate than real-time PCR in the speciation of all five human malaria parasites. The present study also showed a significant increase of the potential fatal P. knowlesi infection in Sabah state as revealed by molecular PCR assays

Chemical changes and optimisation of acetous fermentation time and mother of vinegar concentration in the production of vinegar-like fermented papaya beverage

Fermentation has been long used as a method to produce beverage of various health benefits. In this research, ripe papaya (Carica papaya) was fermented through alcoholic fermentation using Saccharomyces cerevisiae, followed by acetous fermentation using Acetobacter spp. from mother of vinegar, to reduce wastage of this highly perishable Malaysian fruit. The papaya juice was pasteurised prior to the fermentation process. Optimisation of acetous fermentation was carried out using the response surface methodology (RSM) with central composite rotatable design (CCRD). Acetous fermentation time had shown significant effect on all the chemical characteristics while mother of vinegar concentration did not significantly effect on all the chemical characteristics. The vinegar-like fermented papaya beverage which was produced at the optimum point (Fermentation time = 70.80 h and concentration = 40% mother of vinegar) contained 0.37 ± 0.01% reducing sugar, 3.54 ± 0.36% ethanol, 2.46 ± 0.07% acetic acid, 327.89 ± 3.60 mg GAE/ L total phenolic, 2.32 ± 0.17 mg/100 mL ascorbic acid and 52.40 ± 0.23% mg AA/100 mL free-radical scavenging activity. In conclusion, vinegar-like fermented papaya beverage was successfully produced and its chemical compositions changed from papaya juice to wine and vinegar-like beverage with increased bioactive compounds and antioxidative activity