BACKGROUND: Hydroxyapatite is widely used as bone graft substitute. Platelet-rich plasma (PRP) is enriched with growth factors. Hypothetically combination of PRP and hydroxyapatite would augment bone formation effect of hydroxyapatite and widen its application in spinal fusion. OBJECTIVE: To compare new bone formation between hydroxyapatite granules alone; hydroxyapatite granules in combination with PRP; and autograft as the control. METHODOLOGY: First phase of the study dealt with the production of PRP and characterization of platelets by platelet counts and platelet morphology. Second phase involved spinal fusion surgery in twenty-four adult New Zealand white rabbits. All the animals underwent single level bilateral intertransverse process fusion at L5-L6 vertebrae. One side of the animals received either hydroxyapatite granules alone (HA group) or combination of hydroxyapatite granules and PRP (HAPRP group) while the contralateral side received autograft (Autograft group). Four animals each in HA vs. Autograft and HAPRP vs. Autograft were assessed either at 6, 12 or 16 weeks by plain radiograph, computed tomography scan, undecalcified histology, histomorphometry and scanning electron microscopy. Using the histomorphometric data, the mean percentage of new bone areas over the corresponding fusion masses was compared between groups. RESULTS: Mean platelet count in whole blood and PRP were 363 x 103/3/uL (SD 112 x 103) and 1596 x 103/3uL (SD 512 x 103) respectively. Platelets showed minimal morphological changes. Autograft group showed significantly more new bone than HA and HAPRP at 6, 12 and 16 weeks (P=0.004, P<0.001 and P<0.001 respectively) but no significant difference between HA and HAPRP at 6, 12 and 16 weeks (P=0.154, P=0.929, P=0.487 respectively). Autograft, HA and HAPRP groups showed direct contact with new bone but Autograft demonstrated better integration than HA and HAPRP groups. CONCLUSION: Hydroxyapatite granules alone or in combination with PRP could not challenge autograft as bone graft substitute for posterolateral lumbar fusion. [305 words]

Information Not Available

Recently, hepatitis B virus antigen (HBsAg) level has been used as a cheaper marker than molecular methods, not only for indicating active hepatitis B infection but also for predicting the clinical and treatment outcomes. However, data correlating HBsAg level with other diagnostic markers in Malaysia are inadequate to investigate the legitimacy of using HBsAg level as a surrogate or complementary serological marker for chronic hepatitis B (CHB) patients. Therefore, the goals of this study were to quantify HBsAg level in CHB patients and investigate its correlation with immune status (exemplified by peripheral blood lymphocytes’PBL’ subset counts), HBeAg, anti-HBe, viral DNA load, HBV genotypes, and the presence of precore (G1896A) mutations.Methodology:A total of 50 CHB cases and 20 healthy controls were recruited for this cross-sectional study. Serum samples from cases were evaluated for both serological and virological parameters. The HBsAg concentrations for all patients were measured using Roche’s Elecsys HBsAg II assay. The immune status for both study populations were evaluated by estimating the percentage and count of PBL. Viral DNA from all patients’ sera was used for subsequent molecular tests such as PCR assay for HBV DNA detection and genotyping. Real time PCR with SYBR green assay was also performed for viral DNA quantification. Lastly, precore mutants of HBV were detected using high resolution melting analysis. The data were analysed to discover the relationship between HBsAg levels and the other parameters. Results: Viremic CHB patients exhibited narrowly higher mean levels of bilirubin and liver enzymes than the controls. The study of peripheral blood lymphocytes revealed that the percentage of CD4+& CD8+cells were significantly reduced in the patients’ while CD4+/CD8+ ratio has increased. The measurement of HBsAg titers showed that the first group which represents 56% of patients are those with HBsAg levels of >1000 IU/mL, while, second group with <1000 IU/mL represents 44% of CHB patients. Specific viral S small gene DNA was identified in most patients (n=35) and the phylogenetic tree analysis for these samples elucidated that the sequences were classified as genotypes C & B with prevalence rates 54.2% for genotype C and 45.7% for genotype B. In the viral load study, a total of 15 out of 35 patients have a high viral DNA load? 3×106genome copies/mL, while 7 patients were regarded having a low viral load < 3× 106 genome copies/mL. The remaining 13 patients were categorised as undetectable viral DNA load (3× 103 copies/mL). The correlation outcomes showed that a higher number of patients with low-HBsAg level are positive for anti-HBe antibodies. In addition, there is a positive correlation between surface antigen levels with elevated ALT enzyme, and significant positive and negative correlations have been found between HBsAg titres and the percentage of total T and NK cells respectively. The analysis of association between HBV genotypes and HBsAg levels revealed that patients with genotype C have the higher serum level of this protein than genotype B. Other finding has showed no significant relationship between viral DNA copy number and HBsAg level. The precore (G1896A) variants of HBV were detected in only 25% of patients who tested negative for HBeAg but did not illustrate any correlation with HBsAg titres. Conclusion:The overall outcomes presented in this study are conducive to the importance of quantitative HBsAg serum level as a clinical complementary laboratory biomarker for managing and predicting chronic hepatitis B virus infection progression in the Malaysian’ tested patients.

Schizophrenia is a chronic and disabling mental illness with unknown cause and incompletely understood pathogenesis. Evidence from genome-wide association studies (GWAS) and experimental studies had suggested the role of two immune related proteins, the complement C4, coded partly by the C4A gene, and the CUB and Sushi Multiple Domains 1 (CSMD1). However, there was no available report on the association between schizophrenia and DNA methylation of the C4A and CSMD1 genes. Such study is important because DNA methylation is a modifiable factor that can affect candidate genes’ expression and therefore explain the genetic-environment interaction in schizophrenia’s pathogenesis. Both genes also have copy number variation (CNV) which can influence gene expression. This study aims to compare the DNA methylation level and the copy number of C4A and CSMD1 genes between schizophrenia patients and healthy controls, and to evaluate their relationship with schizophrenia psychopathology. A total of 183 schizophrenia patients and 212 healthy controls were included in this comparative cross-sectional study. DNA methylation levels and gene copy number were determined from peripheral blood samples using MethyLightTM analysis and droplet digital polymerase chain reaction (ddPCR) respectively. C4 plasma levels was measured using immunoturbidimetry. Psychopathological data of patients were measured using the Positive and Negative Syndrome Scale (PANSS) and the Personal and Social Performance (PSP) scale. Plasma C4 levels were found to be significantly higher in schizophrenia patients compared to controls (p < 0.001). While C4A DNA methylation levels and copy number were both positively correlated with plasma C4 levels (p < 0.001), there was no significant difference in the two variables between patients and controls. The DNA methylation levels of CSMD1 were significantly lower in schizophrenia patients compared to healthy controls (p = 0.001), but its copy number did not differ significantly between the groups. C4A deletion and higher CSMD1 DNA methylation levels were also associated with lesser positive symptom severity (p = 0.027). In multivariate analysis, both CSMD1 DNA methylation levels and plasma C4 levels were significant predictors for schizophrenia. Overall, the results suggested the potential involvement of DNA methylation of C4A and CSMD1 in schizophrenia pathophysiology, particularly in pathways relevant to the positive symptoms. Since DNA methylation may be reversed, this could be a useful target in for the development of new treatment in the future. Further studies are required to identify the underlying mechanism for these findings.

Hypertension is emerging as the most prevalent risk factor of ischemic heart disease in young adults, but awareness is low in this age group. The prevalence of prehypertension in this population is also high, putting them at higher cardiovascular risk. The pathophysiology of essential hypertension has yet to be fully understood, and epigenetic modifications have been proposed to play some role. To date, very few epigenetic studies were done in young adults with prehypertension and hypertension. The aim of this study was to compare the level of DNA methylation in the promoter of implicated genes in young adults with normotensive blood pressure, prehypertension and hypertension. An observational cross-sectional study was conducted among 240 subjects age 18 to 45 years in Kuantan, Pahang, Malaysia. Eighty subjects were recruited for each blood pressure group; normotension, prehypertension, and hypertension as defined by the Ministry of Health Malaysia Clinical Practice Guidelines 4th edition. MethyLight analysis was performed to determine DNA methylation levels of IL-6, ADD1 and AGTR1 gene promoter in the blood. Differentially methylated genes in prehypertension and/or hypertension group were followed by gene expression study (n = 10 per group). There was no significant difference in IL-6 methylation between hypertensive and normotensive. IL-6 predicted prehypertension in males (p = 0.014), but not females. Hypertensive and prehypertensive males, and prehypertensive females, had lower ADD1 methylation than their respective normotensive counterparts. After adjusting for other covariates, ADD1 methylation predicted prehypertension and hypertension in males (p = 0.002 and p = 0.034 respectively). There was no significant difference in AGTR1 methylation between the three groups in both sexes. There was no significant association between IL-6 and ADD1 methylation level and gene expression level. DNA methylation of IL-6 and ADD1 are independent predictors of prehypertension and/or hypertension in males hence has potential as an adjunct biomarker for risk stratification or disease progression. This is the pioneering study of IL-6, ADD1 and AGTR1 methylation in prehypertensive and hypertensive young adults. Further study to delineate potential mechanisms linking DNA methylation to disease development is warranted.

A variety of evidences of genetic factors had been implicated in schizophrenia but the identification of specific gene has proven to be difficult. Based on the dopaminergic hypothesis in schizophrenia, most of the research conducted has focused on genes regulating dopaminergic function. Accumulating evidences suggest the role of DNA methylation in the patho-aetiology of schizophrenia and there are several confounding factors that may contribute to the DNA methylation. Therefore, the aims of the study are to assess the DNA methylation of COMT, DRD2 and DRD4 genes in the peripheral blood and their associations with the psycho-pathological symptoms, antipsychotic drugs treatment and BMI and the effect on the gene expression. The case control cross sectional study consisted of 138 schizophrenia patients from the Psychiatry Clinic, Hospital Kuantan Ampuan Afzan, Kuantan Pahang and 132 healthy controls from Kuantan district. The genomic DNA samples from the peripheral blood were bisulfite converted. The COMT, DRD2 and DRD4 DNA methylation levels were quantitatively measured by using the MethyLight Taqman® assay and normalized with the ALU reference control to give the percentage methylation ratio. The psycho-pathological symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS). The demographic data were calculated using descriptive statistics while parametric variables were compared using independent samples t-test or analysis of covariance, and the regression analysis was used for prediction. The independent-t test showed significant lower DNA methylation of COMT (p<0.001), DRD2 (p=0.001) and DRD4 (p=0.001) in schizophrenia patients. The lower methylations of each gene were also significant in males and females. There were inverse relationship between the DNA methylation of the dopamine associated genes and psycho-pathological symptoms. COMT, DRD2 and DRD4 DNA methylation were significantly correlated (p <0.002) with, and predictors of the Excitement and the Depressed subdomains of PANSS. In addition DRD4 DNA methylation was highly correlated (p=0.010) and significant predictor of the Disorganization subdomain. There were higher methylations of COMT and DRD2 in typical antipsychotics-treated patients (p?0.05). The overweight BMI schizophrenia patients showed higher COMT and DRD2 DNA methylation (p?0.05). In conclusion, this study strongly support the possible role of COMT, DRD2 and DRD4 DNA methylation could contribute in the patho-aetiology of schizophrenia. The relationship between the DNA methylation of dopamine-associated genes with the psycho-pathological symptoms and the antipsychotics used might indicate the epigenetic role of genes methylation in the manifestation of schizophrenia and their possible role in the mechanisms of action of antipsychotic drugs. The association of the DNA methylation of COMT, DRD2 and DRD4 with BMI support the theories that obesity in schizophrenia is multifactorial and DNA methylation could be one of the contributing factors.

Patients with diabetes are susceptible to develop chronic, nonhealing wounds which cause pain, suffering, and poor quality of life. This, together with high prevalence of delayed wound (15%), increases necessity to find new and more efficient approaches for diabetic wound treatment. Researchers have explored flaxseed oil to expedite in vivo wound healing. Flaxseed oil is known for its anti-inflammatory and antioxidative effects that improve wound healing because the inflammatory process and oxidative damage are implicated in the pathogenesis of diabetic wounds. However, studies utilising flaxseed oil on diabetic animal models are scarce. This study investigates the therapeutic effect of flaxseed oil on wound healing in diabetic animals in 4th, 7th, and 14th day intervals. The study has two phases: the streptozotocin (STZ) diabetes induction phase consisting of 27 male rabbits and the flaxseed oil treatment phase applied to diabetic and nondiabetic animals consisting of 54 male rabbits, in which a full-thickness skin incisional wound (15–17 mm length) were created. They were divided into flaxseed group for diabetic (n=9) and nondiabetic animals (n=9). Positive control (Fucidin cream 2%) group for diabetic (n=9) and nondiabetic animals (n=9), and negative control (nontreated) group for diabetic (n=9) and nondiabetic animals (n=9). The gross wound was monitored using a digital camera and J image software to measure wound length. Flaxseed group diabetic animals group showed regular and approximate smooth edges of the skin wound with organised brightly coloured eschar tissue. All groups have the same days of complete wound closure. However, the wound healing efficiency was higher for flaxseed group diabetic animals (p<0.05) than the control group. The assessment of skin elasticity for flaxseed group nondiabetic animals had the highest viscoelasticity (VE) values with significant differences for three-week intervals. Histological analyses of H&E and Mallory-Trichrome were used to study wound healing. Immunohistochemical evaluations (VEGF and TGF-β) with biochemical analysis (ELISA) of (MMP-2, PDGF-A, and VEGF) protein expression was performed on day 4, day 7, and day 14 of wound healing. The wound healing of the flaxseed group accelerated initially by increasing cellular proliferation (keratinocytes, fibroblast, and endothelial cell) and reducing inflammation via modulation of the protein signalling pathway. In diabetic animals, flaxseed oil enhanced healing by reducing oxidative damage through increased activities of endogenous antioxidants as the flaxseed antioxidant activity was accompanied by up-regulation of pro-fibrotic (TGF-β) gene expression, which triggers fibrogenesis and angiogenesis of wound healing. These mechanisms were more pronounced in flaxseed groups. This study proved that flaxseed oil is a good product for treating diabetic wounds, either alone or combined with biocompatible and biodegradable wound dressing. In conclusion, the results justified that flaxseed oil can be further developed to obtain new and more efficient dressing agent to treat diabetic wounds and other types of skin wounds.

Hypertension seldom occurs in isolation of other risk factors to which it is metabolically linked. Antihypertensive drugs may increase coronary risk by their effect on metabolic risk factors of coronary heart disease. This study was performed to investigate the effects of 2 new antihypertensive drugs, irbesartan and valsartan, which are angiotensin II receptor blockers. A clinical study was carried out to assess the efficacy, clinical acceptability, hypotensive and adverse effect of irbesartan on known metabolic risk factors as well as laboratory parameters in the Malaysian hypertensive populations. Thirty-four mild to moderate hypertensive patients were included in a single blind study over a period of 3 months. Clinic blood pressures were normalized in 76.5% (n=26) of hypertensives patients, and 79.1 % (n=27) of hypertensive patients responded after treatment with 150mg and 300mg daily irbesartan. The proportion of reported adverse events after irbesartan therapy and placebo were 16% (n=8) and 26% (n=l3) respectively. Irbesartan had no significant effect on heart rate and body weight. An extended oral glucose tolerance test (OGTT) was performed at baseline and after 3 months of treatment in 7 hypertensive patients. It was noted that irbesartan had no influence on plasma glucose tolerance and insulin levels. Irbesartan increased plasma basal insulin levels with no effect on glucose levels in hypertensive patients. It also reduced total cholesterol, low density lipoprotein cholesterol and serum uric acid in hypertensive patients. Haematologically, irbesartan reduced erythrocyte count, and increased the mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increased leucocyte counts. Irbesartan decreased aspartate aminotransferase levels. The noninvasive 24-hour ambulatory blood pressure monitoring showed that irbesartan significantly reduced mean 24-hour blood pressures but did not influence blood pressure variability. Irbesartan has an acceptable trough to peak effect (>50%) in hypertensive patients. In study to identify plasma total homocysteine (tHcy) levels, included 41 hypertensive patients (34 of them were receiving irbesartan at baseline) and 54 healthy normotensives. The potential effects of irbesartan on tHcy levels were also evaluated. The 95% confidence interval of tHcy levels for healthy normotensive and hypertensive subjects were 3.62 to 13.30?mol/L and 5.58 to 16.42?mol/L, respectively. Male normotensives had higher tHcy levels than their female counterparts. The tHcy levels were significantly higher in hypertensive patients compared to normotensive subjects. A positive association was observed between tHcy levels and diastolic blood pressure in normotensive subjects. Irbesartan produced significant reduction in tHcy levels after 3-month therapy in hypertensive patients. At baseline, there was an inverse relationship between tHcy and plasma insulin, but no significant correlation was observed after irbesartan therapy. Finally, an in vitro experiment was conducted to study the effect of glucose-induced insulin secretion using isolated rat pancreas by the perfusion technique. Concentration of valsartan used was based on the peak plasma concentration with a single standard oral dose of 80mg daily in human. Valsartan significantly increased insulin secretion at all concentrations (0.164mg/L or 1/10 of the peak, 1.64mg/L or at peak, and 16.4mg/L or 10 times of the peak).

Chronic exposure to inorganic arsenic has been linked with multiple medical conditions, which shifted the use of inorganic to the organic-based herbicide, monosodium methyl arsenate (MSMA). However, with increasing numbers of chronic kidney disease of unknown causes (CKDu), chronic exposure to herbicide is believed to be one of the potential explanation. To date, studies on the effects of organic arsenic exposure on the kidney are limited. Therefore, this study aimed to investigate the effect of chronic oral organic arsenic exposure on the rat’s kidney. Thirty-six Sprague Dawley rats (N=36) were randomly divided into MSMA exposed, and its corresponding control groups for 2-,4- and 6-month, each with six animals per group. The exposed groups were given oral MSMA at 63.20 mg/kg body weight, while control groups received distilled water. At the end of each duration, the serum was collected for the creatinine level. The kidney tissues were harvested for arsenic level measurement, histopathological, immunohistochemistry, real-time PCR analysis and ultrastructural analysis. Genes expressions were done for kidney injury marker gene (KIM-1), oxidative stress genes (Catalase, GSR, NOS1), apoptosis genes (Tp53, Caspase-3 and Caspase-9) and inflammatory genes (Interleukin-6 and Interleukin-8). Serum creatinine was not significantly different between exposed and control groups. Tissue arsenic level was significantly higher in exposed groups as compared to that of the control group. All gene expression markers were downregulated at 2-month and upregulated at 4-month except for Catalase which remained downregulated. At 6-month, only KIM-1, GSR and Caspase-3 remained upregulated. Histological, immunohistochemistry and ultrastructural findings showed chronological changes in the glomeruli and proximal tubules with increased expressions of malondialdehyde (MDA) staining, Caspase-3 and TUNEL staining with the duration of exposure. Therefore, chronic oral exposure to low dose organic arsenic has demonstrated evidence of kidney injury in rats possibly due to oxidative stress.

Introduction: Chlorpyrifos (CPF) is an organophosphate (OP) that is widely used as pesticide in agriculture. Epidemiological studies reported that the incidence of chronic kidney disease (CKD) of unknown cause was increasing among agricultural workers who were exposed to OPs during their working life through dermal contact. However, there is little information on the effects of chronic subcutaneous low dose of OP CPF on kidney in experimental animals. To date, the mechanism of OP-induced kidney damage is not fully elucidated. Objective: The aim of this study was to assess the effects of chronic subcutaneous low dose of OP CPF exposure on the kidney by investigating the structure and function of kidney and to assess the possible mechanisms of OP-induced kidney damage. Methodology: Eighteen male Sprague Dawley rats were randomly divided into three groups, with six rats in each group. Group 1 served as control group, while groups 2 and 3 received subcutaneous vehicle (3% dimethyl sulfoxide + 97% v/v soy oil) and CPF (18.0 mg/kg) respectively, every other day for 180 days. Blood was taken for biochemical analysis while kidney tissues were harvested for histology, immunohistochemistry (IHC) and selective gene expression. Cystatin C, acetylcholinesterase (AChE), advanced glycation end products (AGEs) and malondialdehyde (MDA) levels were measured using quantitative sandwich enzyme immunoassay. Results: Biochemical parameters (urea, creatinine, uric acid, glucose), cystatin C, AGEs and MDA levels were significantly increased (p < 0.05), while AChE activity and electrolytes levels were significantly decreased (p < 0.05) in the CPF-exposed rats. Structural damage of kidney, including diffuse global glomerular hypercellularity and diffuse necrosis of proximal tubular cells were observed in CPF-exposed kidney. IHC revealed strong immunostaining of polyclonal anti-AGEs in glomeruli and polyclonal anti-MDA in the proximal tubular cells of CPF-exposed rats. The expression of genes involved in glucose metabolism (Ager), oxidative stress (Sod3, Cat, Gsr, Pon1 and Nos2) and cell death pathways (Cycs, Casp3, Casp, 8, Casp, 9, Ripk1, Ripk3, Cst3, Havcr1 and Lcn2) showed downregulation trend. Conclusion: Chronic subcutaneous low dose of CPF caused renal dysfunction as evidence by increased endogenous glomerular filtration markers and reduced electrolytes levels and structural kidney damage. The downregulation of these genes could be due to selective exhaustion of pathways that were persistently activated during the prolonged chronic OP-mediated injury. In brief, chronic subcutaneous low dose of CPF caused nephrotoxicity. The process could be facilitated by the effects of CPF on glucose metabolism and oxidative stress.

Hypertensive disorders of pregnancy (HDPs) contribute to a significant percentage of maternal and foetal morbidity and mortality worldwide. Despite the normalisation of blood pressure postpartum, women with a history of HDPs have an increased two-to-four-fold risk of developing cardiovascular diseases (CVDs) later in life. One of the aetiologies of CVDs is endothelial dysfunction. We hypothesised that the transient high blood pressure during HDPs leads to persistent and ongoing endothelial dysfunction (ED) and, ultimately, the development of CVDs in women. This study aimed to explore the effects of high blood pressure during pregnancy through histopathological, biochemical, immunohistochemical (IHC), and ultrastructural studies at one month postpartum. Twenty-four female Sprague-Dawley (SD) rats were assigned to four groups (n = 6), comprising two treatment groups administered with N?-Nitro-L-Arginine Methyl Ester Hydrochloride (L-NAME) and two control groups. All rats were sacrificed on Day 30 postpartum. The mesenteric arteries (resistance arteries) were harvested and preserved for histopathological and ultrastructural studies. Blood was taken for the biochemical study of nitric oxide (NO) and endothelin-1 (ET-1), and their concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The endothelin-1 A receptor (ETAR) and endothelin-1 B receptor (ETBR) expression of the resistance arteries were measured by immunohistochemical studies. During the postpartum period, the mean concentrations of ET-1 and NO were not significantly altered in all groups, and there were no significant changes in the mean immunoreactivity of the ETAR and ETBR for the tunica intima and media. For quantitative studies, the mean media-to-lumen ratio, and the endothelial cell counts per length ratio were preserved between the control and treatment groups. Although the mean nucleus-to-cytoplasmic ratio or internal elastic lamina (IEL) thickness remained unaltered between the control and treatment groups, the ultrastructural examination using a transmission electron microscope revealed remarkable ultrastructural changes in the resistance arteries that were not visible by histopathological study. The endothelial cells of the pregnant + L-NAME (PL) group exhibit irregular nuclei, discontinuous cytoplasmic boundaries, numerous abnormal vacuolization, dilatation of subendothelial space (SES), fragmented IEL, and abundant extracellular matrix (ECM) substances below IEL. In conclusion, the endothelium of the resistance arteries of hypertension-induced pregnant rats showed substantial evidence of ultrastructural changes after postpartum. Despite there being no further insult, it indicates that there is an initial pathology for ED due to high blood pressure during HDPs. This may result in the later development of CVDs in women.

Globally, snakebite cases are estimated to be around 5 million annually affecting mainly the residents of poorer counties like Africa and Asia, and in 2009 WHO has categorised it as a ‘neglected tropical disease’. Currently the standard treatment for snake envenomation is the use of anti-snake venom (ASV) therapy. However this is expensive and not readily available in smaller hospitals in the developing world. Herbal medicine has been and is still in use in some cultures for the treatment of snakebite and one such plant is Tamarindus indica. This plant is found in many countries where snake envenomation is also prevalent. This study was conducted to evaluate the potential of using T. indica seed extract (TSE) to inhibit the effects of snake venom of three snakes; namely Naja kaouthia, Ophiophagus hannah and Daboia russelli. The testa of tamarind seed was used and it underwent ethanolic soxhlet extraction to obtain TSE. The inhibition of the activity of the following enzymes i.e phospholipase A2 (PLA2), proteinase and phosphomonoesterase (PME) in vitro by the three snake venoms with TSE was studied. SDS-PAGE experiment was conducted to observe the effects of TSE on venom proteins. In vivo acute subcutaneous (SC) toxicity of TSE in ICR mice was conducted. Study on the inhibition of lethality was conducted on each of the three snake venoms when SC TSE was injected into mice. Venom concentration and site were fixed but TSE concentration, time and site of injection were manipulated. Findings from venom enzymatic inhibition studies showed that, TSE was able to significantly reduce (p<0.05) all three venom enzymatic activities i.e PLA2, proteinase and PME. SDS-PAGE experiment showed that venom protein bands were disrupted when venom reacted with TSE. No signs of toxicity were observed over a period of 4 weeks when mice were exposed to SC TSE 60 mg/20 g body weight except for skin ulcers. Histological examination on liver, both kidneys and skin at the site of SC injection showed no changes compared to the control group injected with SC distilled water. TSE was able to increase the survival rate of ICR mice when exposed to each of the three snake venoms regardless of the site of injecting SC TSE. Mice injected with N. kaouthia or D. russelli venom, had increased 24 hour survival rate when SC TSE was given at 15 minutes; and of mice injected with O. hannah venom the 24 hour survival rate increased with higher TSE concentration when given sooner. In conclusion, SC TSE was safe to be injected up to 60mg/20 g and has the potential to delay the effects of venom from N. kaouthia, O. hannah and D. russelli.

Ticlopidine is used as an anti-platelet drug in patients with ischaemic heart disease. An in vitro study suggested that ticlopidine inhibited CYP2D6 and the widely used antianginal metoprolol is metabolized by this polymorphic enzyme. The objective of this study to investigate the effect of ticlopidine treatment in patients maintained on chronic metoprolol therapy. The study was approved by the Ethics Committee of International Islamic University Malaysia (IIUM) and strictly adhered to Malaysian Good Clinical Practice (GCP) guidelines. This was an open labelled Case Controlled Study where all the patients were screened for the inclusion and exclusion criteria. CYP2D6 genotyping were performed for *3,*4,*5,*6,*9,*10, *14, *17 and duplication. Two weeks after the screening visit, blood for metoprolol was taken at timed intervals together with serial measurement of blood pressures and heart rates. Subsequently the patients were given a standard dose of ticlopidine 250 mg twice daily for a period of one month. At the end of study period, blood for metoprolol was repeated together with serial measurement of blood pressures and heart rates. Eighty seven patients completed the study. The frequency of predicted Poor Metabolizer (PM) was low at 2.6%, where both patients had homozygous *4/*4 and the majority (47.8%) carried the allele *10, predicted Intermediate Metabolizer (IM). After ticlopidine treatment, there were increasing trend within the metoprolol pharmacokinetic parameters among the different predicted phenotypes and different allele variations. Plasma Metabolic Ratio was significantly different (p< 0.05) between phenotypes and allele variations. The two Poor Metabolizers (PM) patients presented with bradycardia even at doses of 25 mg and 50 mg twice daily. There were wide variations among the CYP2D6*10 allele group for metoprolol pharmacokinetic parameters, suggesting the presence of a subgroup population that may overlap features with the Poor Metabolizer group. However, there were no significant change for both pre and post ticlopidine for blood pressure control and heart rate. After ticlopidine, there was a neutrophil count reduction in 32 patients and 6 patients had neutropenia with neutrophil count less than 2.5 x 10³ / μl. Concurrent use of ticlopidine with metoprolol may subject patients who are poor CYP2D6 metabolisers to have exaggerated response to beta blockade and blood dyscrasias may occur frequently.

Hepatitis C infection in haemodialysis (HD) centres is still of utmost global concern to health care systems. Studying the molecular features of the virus throws light on its epidemiology and behavior in those undergoing prolonged HD. In this study, hepatitis C virus (HCV) infecting HD patients in Pahang were characterized and the prevalence of the various genotypes and subtypes determined and the full-length genome of the most prevalent genotype was elucidated. Viral RNA was extracted from sera of 40 HD patients. The molecular technique of reverse-transcription polymerase chain reaction (RT-PCR) was used to detect and amplify segments of the viral RNA at the 5’untranslated region (UTR) and the nonstructural 5B region (NS5B). These regions were well conserved among HCV genotypes thus making them useful for both virus detection and genotyping purposes. The base sequences of the amplicons were determined and later employed in phylogenetic analysis for genotype assignment of the isolates. The sequence pattern (SP) and the conformational features of domain III 5’UTR were also evaluated. The overall prevalence of HCV viremia in HD centres was 8.3 %, including one acute infection that occured during the course of study. For 33 of the 40 viremic patients, the HCV 5’UTR and NS5B base sequences were successfully determined. Five patients (15.2 %) were shown to be infected by more than one genotype. In the remaining 28 patients, genotypes 3, 1, 4, and 6 occurred in 19 (67.9 %), seven (25.0 %), one (3.6 %), and one (3.6 %) of them respectively. Four sequence patterns (SPs) were determined in domain III 5’UTR of genotype 3, a finding unique to this genotype. Overall, the secondary structure of the domain was not affected by nucleotide changes in the SP region. The complete base sequence of two genomes of the predominant subtype 3a isolates (MAL 22 & MAL 43) were elucidated and found to be consisting of 9430 and 9429 nucleotides. Genotype 3a HCV genome contained a long open reading frame capable of encoding a sequence of 3010 amino acid residues. In the phylogenetic analysis, both isolates clustered with known HCV-3a isolates (NZL1, K3a & CB), while it diverged from other genotypes by 47.1 - 41.8 %. Three hypervariable regions of the isolates were also revealed and described. The predominance of genotypes 3 and 1 was not unique to HD centres in Pahang as it was reported in normal population in Malaysia. Both the complete viral nucleotide sequence and the amino acid sequence of the coding region of the HCV genome provided the foundation data for future HCV studies in Malaysia including studies on epidemiology, novel diagnostic approaches and antiviral therapies. Despite the known high risk of chronic hepatitis C infection in HD patients, these infections, whether overt or occult and of single or mixed genotypes, were usually clinically silent. Thus, investigational viral nucleic acid based tests on HCV patients was recommended to be done periodically to monitor for the occurrence of these asymptomatic and sometimes even sero-negative infections.

Acute myocardial infraction (AMI) is the most common clinical manifestation of coronary artery disease (CAD). Young age is no longer considered a protective factor since the incidence of young adults with AMI is increasing. Hypertension is an important risk factor for CAD in young adults. Prehypertension without proper management is also associated with an increased risk of CAD. Hence, the identification of CAD biomarkers in young hypertensive and prehypertensive adults is necessary to improve risk stratification of premature AMI in these cohorts. The main objective of this study was to compare protein expression profiles of young adults with AMI to control subjects for the identification of proteins (candidate biomarkers) that are differentially expressed in AMI patients. This study also aimed to determine the plasma concentrations of the candidate biomarkers in young adults with normotension, prehypertension, hypertension and AMI and evaluate the relationship between AMI and potential CAD biomarker/s in young hypertensive and prehypertensive subjects. This study comprised of two phases; discovery and verification. In the discovery phase, proteins in the pooled plasma samples from young male adults (10 AMI patients and 10 controls) aged 18 to 45 years were separated by two-dimensional gel electrophoresis (2-DE). The protein spots that were differentially expressed in AMI patients relative to the controls were identified via matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. In the verification phase, the plasma concentrations of the identified proteins were measured using enzyme-linked immunosorbent assay (ELISA) in 40 plasma samples of control, prehypertensive, hypertensive and AMI groups. In the discovery phase, haptoglobin (Hp), apolipoprotein AI (Apo AI) and apolipoprotein AIV (Apo IV) were significantly upregulated in AMI patients in comparison to the controls (p < 0.05). Meanwhile in the verification phase, the plasma concentration of Hp was significantly higher in AMI patients in comparison to the control, prehypertensive and hypertensive subjects (290.63±99.90 vs. 170.02±108.11 vs. 175.05±108.11 and vs. 208.47±112.97 ng/ml, p < 0.006) respectively. The plasma concentrations of Apo AI and Apo AIV were also elevated in AMI patients, yet the increases were not significant compared to the other groups (p > 0.05). Plasma concentration of Hp was significantly associated with young AMI (OR: 1.019, 95% CI: 1.006-1.033, p = 0.003) after adjusting for other known CAD risk factors. There was also a significant association between AMI and plasma concentration of Hp in hypertensive and prehypertensive subjects (OR: 0.985, 95% CI: 0.973-0.997, p = 0.017 and OR: 0.981, 95% CI: 0.969-0.993, p = 0.002) respectively, independent of other known CAD risk factors. Plasma Hp concentration was significantly correlated with high sensitivity C-reactive protein hs-CRP (r = 0.370, p < 0.001). In Conclusion, consistent upregulation of Hp in discovery and verification phases reflect its potential role as a biomarker of CAD in young adults. Hp is also a potential CAD biomarker that could be utilized as AMI predictor in young adults with hypertension and prehypertension.The significant correlation between Hp and hs-CRP indicates the potential role of these proteins as inflammatory markers in the establishment of CAD in young adults.

Acute myocardial infarction (AMI) is a severe form of coronary heart disease where Malaysians are getting AMI at younger age compared to well-developed countries. MicroRNAs (miRNAs) are implicated in AMI pathogenesis, but no study looked at their profiling or involvement in young population. The present study aims to profile the miRNAs expressions in healthy controls (aged 18 to 45 years), young AMI (YAMI) (aged ≤ 45 years), and mature AMI (MAMI) (aged ≥ 46 years) patients with matching criteria and to determine the effect of the dysregulated miRNAs on the target mRNAs as well as the pathways involve in the pathogenesis of AMI. This study was conducted on twenty Malay males for each group in Kuantan, Pahang. Total RNA was extracted from plasma and the miRNA expression profiling was carried out on the BGISEQ500 SE50 sequencing platform with BGI sequencing libraries. The sequence data were analyzed using Gene Ontology (GO) to determine the role of the differentially expressed genes, followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for identification of the biological pathways in YAMI against MAMI. The top six dysregulated miRNAs identified during sequencing were validated using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) between the groups. ANOVA and unpaired T-test were used to analyze the differences of miRNAs and gene expression between the three groups. This study revealed that majority AMI patients were smokers, where YAMI patients had higher BMI, SBP, DBP and TG while MAMI patients had higher FBG than the rest of the group. A total of 1599 miRNAs were differentially expressed in AMI (YAMI and MAMI) patients compared to healthy controls, where 1288 were upregulated and 311 were downregulated (FDR ≤ 0.001). However, when YAMI patients were compared to MAMI patients, 1497 miRNAs were found to be dysregulated, of which 1090 miRNAs were upregulated, and 407 miRNAs were downregulated (FDR ≤ 0.001). The top ten upregulated miRNAs were miR-552, miR-4446-3p, miR-432-5p, miR-548j-5p, miR-219, miR-982, miR-181a-2-3p, miR-654-5p, miR-58 and miR-548k; while the top ten downregulated were miR-16-5p, miR-1064, miR-431-5p, miR-790 miR-1177, miR-201, miR-105, miR-518, miR-419 and miR-1103. This study also discovered ten novel miRNAs: miR-4446-3p, miR-982, miR-58, miR-548k, miR-1064, miR-790, miR-1177, miR-201, miR-419, and miR-1103. The validation of the top six dysregulated miRNAs between YAMI and MAMI patients revealed the upregulation of miR-423-5p by 2.08-fold (p = 0.040) and downregulation of miR-431-5p by 33.90-fold (p = 0.034), and miR-378a-5p by 34.61-fold (p = 0.040). For these 1497 differentially expressed miRNAs, 34,195 target genes were predicted by GO analysis. The functional analysis demonstrated 11,199 GO terms found to be involved in biological processes, 12,012 in cellular components, and 10,984 in molecular functions were significantly enriched (p < 0.05). The target genes that were mapped to the signal transduction pathway in KEGG revealed 346 classes were enriched. In conclusion, miRNAs are differentially expressed between young and mature AMI, ten of which are novel. Three biological pathways, ascorbate and aldarate metabolism, collecting duct acid secretion and glycosaminoglycans biosynthesis – heparin sulfate/heparin were identified but their involvements in the regulatory mechanisms on gene expression in Young AMI need further evaluation.

Decreased cerebral blood supply to the brain can generate a condition of chronic cerebral hypoperfusion, which is one of the physiopathological mechanisms of neuronal degeneration and cognitive impairment in neurodegenerative disorders including Alzheimer’s disease. Trigonella foenum graecum and Eurycoma longifolia Jack have been shown to have antioxidant and anti-inflammatory activities. Therefore, the aim of the current study was to evaluate the potential neuroprotective effect of ethanolic extract of Trigonella foenum graecum seeds (FS) and methanolic extract of Eurycoma longifolia roots (TA) on chronic cerebral hypoperfusion using rat animal model. Chronic cerebral hypoperfusion was induced by permanent bilateral ligation of the common carotid arteries in male Sprague–Dawley rats. The experimental groups were divided into four groups: a sham-operated group, a two-vessel occlusion (2VO) group, a 2VO group that was administered orally with the FS extract (100 mg/kg/day), and a 2VO group that was administered orally with the TA extract (100 mg/kg/day) from 3 days before the date of 2VO surgery and continued until the end of the 8th postoperative week. Spatial memory performance was assessed by the Morris water maze test. Serum malondialdehyde (MDA) content and superoxide dismutase (SOD), glutathione (GSH) activities, C-reactive protein (CRP) concentration were measured. The ultrastructural changes of the hippocampal area (CA1) were observed by transmission electron microscopic (TEM). Chronic cerebral hypoperfusion rats resulted in spatial memory impairments. This behavioural dysfunction was accompanied by decreasing SOD and GSH activities, increasing MDA content and increasing concentration of inflammatory marker (CRP) in serum. Similarly, ultrastructural neurodegenerative changes were observed in CA1 area of the 2VO group, which showed necrosis and apoptosis of pyramidal neurons, astrocytic degeneration with microgliosis, and thickening of endothelial basement membrane of hippocampal capillaries. Oral administration of FS extract significantly improved the memory impairment, enhanced antioxidant enzyme activities, decreased the content of MDA and the CRP levels to their normal levels as well as less ultrastructural changes in CA1 area of hippocampus. On the other hand, TA extract treatment showed a slight preservation of cognitive function, oxidative status and ultrastructural changes in CA1 region following chronic cerebral hypoperfusion. The potential activity offered by FS showed neuroprotective effect that may be beneficial in cerebrovascular type dementia. TA extract showed lesser neuroprotective effect. Further studies may be done to investigate the underlying mechanism.

Nigella sativa seeds oil (NSO) and Murraya koenigii leaves (MKL) demonstrated robust antioxidant and anti-inflammatory activities in vitro and in vivo. Two vessel occlusion surgery (2VO) is an established rat model of Alzheimer’s disease (AD) which causes chronic cerebral hypoperfusion resulting in oxidative stress and neuroinflammation. These events lead to neurodegenerative brain changes especially within CA1 region of the hippocampus where spatial memory neurons are situated. This experimental intervention study was designed to assess the potential neuroprotective effect of the two herbal plants extracts when given orally to experimental animals ten days before 2VO surgery and continued until the tenth week post 2VO. The assessment was based on cognitive, histopathological and electron microscopical brain tissue analyses. 80 rats were equally divided into 4 main groups namely sham control, 2VO, NSO+2VO and MKL+2VO groups. Each one of them was further subdivided according to the experimental protocol of Morris water maze (MWM) test into long-term memory (LTM) subgroups which underwent acquisition MWM trials before 2VO and were retested for LTM performance on the 10th postoperative week, while the other subgroups were only introduced to MWM during the 10th postoperative week. These subgroups were tested by short-term memory (STM) and working memory test (WMT) protocols in a row. 2VO induced significant LTM, STM and learning impairment in 2VO rat group as compared to sham control as well as NSO+2VO groups, whereas the attenuation of 2VO induced memory impairment achieved by MKL treatment was significantly lower than that of sham control and NSO+2VO groups. The brain tissue analyses included histopathological as well as Transmission Electron Microscopy (TEM) examinations. Light microscope histopathology revealed significantly higher number of viable hippocampal cells in sham control and NSO treated rats groups as compared to untreated 2VO and MKL treated groups. Simultaneously, ultrastructural neurodegenerative changes were observed in CA1 hippocampal subfield of 2VO group which comprised necrosis and apoptosis of pyramidal neurons, astrocytic degeneration with microgliosis and thickening of endothelial basement membranes of hippocampal capillaries. These changes were substantially less demarcated in NSO treated group and were almost completely absent in sham control group. However, a great deal of similarity in ultrastructural deformities was found between untreated 2VO and MKL+2VO groups. It can be concluded that NSO, by virtue of its antioxidant and anti-inflammatory activity, was capable of preventing hippocampal neurodegeneration induced by 2VO, while MKL extract was only able to exert a modest preservation of memory without a neuroprotective effect on CA1 hippocampal neurons after cerebrovascular hypoperfusion initiated by 2VO surgery.

The search for new antimalarials is a continuing effort as the resistance to current antimalarial drugs occur one after another. Thus this study aims to discover a potential antimalarial in the Malaysian local plant. Plectranthus amboinicus is selected as a candidate for antimalarial study. There were four phases involved throughout this study. Phase one focused on the phytochemical screening of ethanolic extract and essential oil of P. amboinicus leaves. The phytochemical screening of the extracts was conducted according to the qualitative phytochemical screening test and Gas Chromatography Mass Spectrometry (GC-MS). Phytochemical screening revealed the presence of flavonoids (in the crude ethanolic extract), carvacrol (85.14 %), thymoquinone (1.65 %), terpinen-4-ol (0.7 %), octenol (0.62 %), thymol (0.23 %), and dioctylazelate (0.15 %) (in the essential oil). Phase two of the study focussed to evaluate the toxicity status of the experimenting extracts prior to conducting antimalarial study on mice infected with Plasmodium berghei. Acute oral toxicity test was conducted according to the OECD guidelines for the testing of chemicals, acute oral toxicity, limit test-acute toxic class method sections 423. Toxicity study with crude ethanolic extracts of P. amboinicus leaves revealed no toxic effect on the mice administered. The acute oral LD50 was found to be above 5000 mg/kg suggesting it to be safe for consumption. Testing with the essential oil also revealed no signs of toxicity and mortality although histopathology results showed the presence of glomerular apoptosis in kidney and evidence of central venous dilation in the liver. Thus, the LD50 was suggested as above 2000 µL/kg. Phase three focused to determine the antimalarial properties of the crude ethanolic extract and essential oil of P. amboinicus leaves. There were three important antimalarial tests that were performed on both the extracts prepared, namely prophylactic test, suppression test and curability test. An antimalarial study on the crude ethanolic extract showed no reduction of parasitemia of mice infected with P. berghei was seen in the suppressive and curative test, but pronounced reduction of parasitemia in the prophylactic test. The best dose of ethanolic extract was found to be 400 mg/kg which reduced parasitemia by 90.74 %. In contrast, the essential oil of P. amboinicus leaves not only possess some potential as a prophylaxis agent, but also a curative agent, but with a less parasitemia reduction compared to the ethanolic extract. The best prophylaxis dose was found to be 1000 µL/kg with 58.26 % reduction in parasitemia, while the best curative dose was also 1000 µL/kg with a reduction of parasitemia of 65.38 %. Similar to crude extract, no reduction of parasitemia was found in the suppression test of the essential oil. Phase four of the study focused on prophylactic test of partitioned fractions of crude ethanolic extract since the extract showed better reduction of parasitemia compared to an essential oil. The extract was partitioned with 3 different polarities of solvent to yield hexane, ethyl acetate and butane fractions. The prophylactic test on the partitioned fractions of the selective crude extract revealed hexane fraction (with 85.54 % chemo suppression) as the best prophylaxis agent, followed by the butane fraction (with 56.56 % chemo suppression) and ethyl acetate fraction (with 47.72 % chemo suppression). In all fractions 400 mg/kg was the most effective dose as prophylaxis agent. Based on these results, it can be concluded that crude extract shows better prophylaxis effect compared to fraction. P. amboinicus leaves extract shows promise as a new potential antimalarial candidate

Calcium phosphate is an ideal bone substitute material that is widely used for bone repair due to its excellent biological properties including biocompatibility and osteoconductivity. In order to improve the properties of calcium phosphate materials for clinical use, a new injectable self-hardened synthetic bone cement (Osteopaste) was developed. Osteopaste consists of tetra-calcium phosphate (TTCP) and tricalcium phosphate (TCP) powder. It was intended for the treatment of bone fracture or reconstruction of bone defects. The objective of this study was to compare bone formation between Osteopaste and commercialized synthetic bone grafts; JectOS (calcium phosphate) and MIIG-X3 (calcium sulphate) at three different assessment periods. The first phase of the study was to establish the critical size defect in New Zealand White rabbit model. The second phase involved the implantation of Osteopaste, JectOs and MIIG-X3 in critical size defects. Thirty-nine New Zealand White rabbits were divided into four groups (Osteopaste, JectOs, MIIG-X3 and sham). Each group was further divided into three subgroups according to the assessment period either at 6, 12 or 24 weeks. Each subgroup consisted of four rabbits except the sham group which consisted of only one rabbit. A critical size defect of approximately 4.5 mm (width) X 9.0 mm (length) was created at the proximal tibial metaphysis of rabbit's right leg and then implanted with either Osteopaste, JectOs or MIIG-X3. At each assessment period, plain radiograph and computed tomography (CT) scan were performed before the animals were sacrificed for undecalcified histology, histomorphometry and scanning electron microscopy assessments. Using the histomorphometric data, the mean percentage of new bone areas and the length of unbridged defects were compared between groups. In this study, a simple and safe method for performing critical size defect at proximal tibial metaphysis was established. The Osteopaste group exhibited radiographic density in between JectOS and MIIG-X3. The critical size defect in Osteopaste group was bridged by new bone at 12 weeks. In MIIG-X3 group, the defect was bridged at 24 weeks whereas in JectOS group, the defect was not bridged at all assessment periods. New bone area was the largest in MIIG-X3 group followed by Osteopaste and JectOS groups. Osteopaste had formed direct bonding with host bone without intervening soft tissue compared to JectOS and MilG-X3. There were significant differences in new bone area percentages between Osteopaste, JectOs and MIIG-X3 at 6, 12 and 24 weeks post-surgery (P<0.0001 ). In conclusion, the performance of Osteopaste to promote new bone formation is in between JectOS and MIIG-X3.