Two single nucleotide polymorphisms of PDLIM5, rs7690296 and rs11097431, were genotyped using Mass-Array SNP genotyping by Sequenom technology in 244 bipolar disorder patients, 471 schizophrenia patients, and 601 control individuals who were Malay, Chinese, and Indian ethnic groups in the Malaysian population. A significant association was observed in allele frequency between the rs7690296 polymorphism and bipolar disorder in the Indian ethnic group [P=0.02, adjusted odds ratio (OR) 0.058, 95% confidence interval (CI) 0.36-0.93]. A significant association was also observed between the rs7690296 polymorphism and schizophrenia under the recessive model for both Malay (P=0.02, adjusted OR 1.86, 95% CI 1.12-3.10) and Indian (P=0.02, adjusted OR 1.92, 95% CI 1.10-3.37) ethnic groups. However, no association was detected between the rs11097431 polymorphism either with bipolar disorder or with schizophrenia. Therefore, it can be deduced that the nonsynonymous rs7690296 polymorphism could play an important role in the pathophysiology of both bipolar disorder and schizophrenia.
Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.
SSRIs are known for their sexual side-effects with a variable rate of sexual dysfunction (SD). 5HT2A (rs6311) single nucleotide polymorphism (SNP) was found to have significant association with SD. The purpose of this study was to determine the prevalence of female SDD, its clinical correlates and association with 5HT2A (rs6311) SNP in patients with major depressive disorder (MDD) treated with SSRIs.
Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.
Alzheimer's disease (AD) as a neurodegenerative brain disorder is the most common cause of dementia. To date, there is no causative treatment for AD and there are few preventive treatments either. The sphingosine-1-phosphate receptor modulator FTY720 (fingolimod) prevents lymphocytes from contributing to an autoimmune reaction and has been approved for multiple sclerosis treatment. In concert with other studies showing the anti-inflammatory and protective effect of FTY720 in some neurodegenerative disorders like ischemia, we have recently shown that FTY720 chronic administration prevents from impairment of spatial learning and memory in AD rats. Here FTY720 was examined on AD rats in comparison to the only clinically approved NMDA receptor antagonist, Memantine. Passive avoidance task showed significant memory restoration in AD animals received FTY720 comparable to Memantine. Upon gene profiling by QuantiGene Plex, this behavioral outcomes was concurrent with considerable alterations in some genes transcripts like that of mitogen activated protein kinases (MAPKs) and some inflammatory markers that may particularly account for the detected decline in hippocampal neural damage or memory impairment associated with AD. From a therapeutic standpoint, our findings conclude that FTY720 may suggest new opportunities for AD management probably based on several modulatory effects on genes involved in cell death or survival.
Approximately 30% of epilepsy patients do not response to antiepileptic drugs (AEDs). The functional SCN1A IVS5N+5 polymorphism may play a role in response to some AEDs. The purpose of this study was to examine this hypothesis in a cohort study of Malaysian and Hong Kong Chinese epilepsy patients on sodium valproate (VPA) monotherapy and in a meta-analysis.
The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.
Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase.
A 46-year-old man presented with a history of passing bright red blood per rectum over the last one month. He also had on and off diarrhea with visible mucus in the stool for two months' duration. Further history was unremarkable, and physical examination revealed hemorrhoids which were subsequently banded. A colonoscopy was arranged in view of the prolonged diarrhea whereby an edematous and swollen ileocecal valve was seen. This was shown to be due to Trichuris trichiura infection, confirmed on histopathological examination of biopsies taken from the site. The patient was started on oral albendazole treatment and has been asymptomatic on latest followup. This case illustrates an accidental finding of T. trichuria infection on colonoscopic examination, which was done to investigate the patient's prolonged diarrhea.
Several studies demonstrated a link between ABCB1 gene variants and the response to treatment in epilepsy, but the results have been inconclusive. Here, we performed the first haplotype meta-analysis to examine the association of haplotypes of ABCB1 common variants with the response to treatment in epilepsy.
Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
Non-secretory multiple myeloma (NSMM) is a rare variant of the classic form of multiple myeloma (MM). In NSMM, no monoclonal gammopathy can be detected in serum or urine by conventional techniques, making the diagnosis more difficult. We describe a 71-year-old man who had been diagnosed and treated for granulocytic sarcoma one year prior to his recent problems of progressive low-back pain of two months duration. Skeletal X-rays showed diffuse osteolytic lesions with multiple pathological fractures but there was no monoclonal gammopathy in the serum or urine. The biopsy of the lytic lesion on the upper part of the femur showed infiltration by abnormal plasma cells. A diagnosis of NSMM was made and he was treated with chemotherapy. The early diagnostic difficulty and the challenges faced regarding the case are discussed.
Insect larvae and adult insects found on human corpses provide important clues for the estimation of the postmortem interval (PMI). Among all necrophagous insects, flesh flies (Diptera: Sarcophagidae) are considered as carrion flies of forensic importance. DNA variations of 17 Malaysian, two Indonesian and one Japanese flesh fly species are analysed using the mitochondrial COI and COII. These two DNA regions were useful for identifying most species experimented. However, characterisation of the species was not sufficiently made in the case of Sarcophaga javanica. Seventeen Malaysian species of forensic importance were successfully clustered into distinct clades and grouped into the six species groups: peregrina, albiceps, dux, pattoni, princeps and ruficornis. These groups correspond with generic or subgeneric taxa of the subfamily Sarcophaginae: Boettcherisca, Parasarcophaga, Liosarcophaga, Sarcorohdendorfia-Lioproctia, Harpagophalla-Seniorwhitea and Liopygia. The genetic variations found in COI and COII can be applied not only to identify the species of forensic importance, but also to understand the taxonomic positions, generic or subgeneric status, of the sarcophagine species.
Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.
OBJECTIVE: Written action plans are regarded as an important part of asthma self-management education and yet they may not be understood by those with limited literacy skills. This study was designed to produce an understandable pictorial asthma action plan.
METHODS: With advice from a group of doctors and nurses a "standard" written action plan was translated by a medical artist into a series of pictorial images. These were assessed using the techniques of guessability and translucency by a series of adults attending a specialist asthma clinic in London and the same process was subsequently used to assess comprehensibility of the images and plans amongst a group of Somalis living in Manchester, UK and Malaysians in Seremban, Malaysia.
RESULTS: Guessability testing showed that the majority of pictograms were well understood by each of the study groups. Translucency testing revealed close agreement with intended meaning for the majority of the images. One image, depicting extra use of reliever medication scored less well in all populations; two other images scored less well in the Somali and Malaysian groups and reflect less use of certain inhaler devices in other countries. The overall plan was well understood by all patients who were able to adequately recount the appropriate actions to take in different clinical scenarios.
CONCLUSION: We have developed a pictorial asthma action plan understandable by 3 different populations of patients with asthma.
PRACTICE IMPLICATIONS: Pictorial representations have been shown by other studies in other situations to be an effective method of reinforcing the spoken word. The pictorial asthma action plan developed for this study has been shown to be comprehensible, personalised to the individual in the usual fashion. It is now suitable for further evaluation in clinical practice.
Study site in Malaysia: Asthma clinic, Hospital Tuanku Jaafar, Seremban, Negeri Sembilan, Malaysia
This study is a result of an analysis of free and conjugated phytoestrogens daidzein, genistein, daidzin, genistin and coumesterol in human cord blood plasma using LCMS. Cord blood was collected from urban and rural populations of Malaysia (n=300) to establish a simple preliminary database on the levels of the analyzed compounds in the collected samples. The study also aimed to look at the levels of phytoestrogens in babies during birth as this may have a profound effect on the developmental process. The sample clean up was carried out by solid-phase extraction using C18 column and passed through DEAE sephadex gel before analysis by LCMS. The mean concentrations of total phytoestrogens were daidzein (1.4+/-2.9 ng/ml), genistein (3.7+/-2.8 ng/ml), daidzin (3.5+/-3.1 ng/ml), genistin (19.5+/-4.2 ng/ml) and coumesterol (3.3+/-3.3 ng/ml). Distribution of phytoestrogen was found to be higher in samples collected from rural areas compared to that of urban areas.
A 16-year-old student presented with a 4-week history of progressive shortness of breath, loss of appetite, and occasional blood-tinged sputum. The chest X-ray revealed massive right-sided pleural effusion with cardiomegaly. An echocardiogram revealed a large pericardial mass with massive pericardial effusion. Subsequent computed tomography of the thorax revealed a large heterogeneous mass in the right lung with extension into the pericardium. Lung biopsy revealed primitive neuroectodermal tumor (PNET) with small round blue cells, Homer-Wright rosettes, and CD99 positivity. We discuss pericardial metastases of PNET and its implication in this patient.
Cyclosporine is a substrate of cytochrome P-450 3A (CYP3A) subfamily of enzymes and characterized by a narrow therapeutic range with wide interindividual variation in pharmacokinetics. A few single-nucleotide polymorphisms detected in CYP3A genes have been shown to correlate significantly with the CYP3A protein expression and activity. We therefore postulated that these polymorphisms could be responsible for some of the interindividual variation in cyclosporine pharmacokinetics. The objective of our study is to determine correlation if any between single-nucleotide polymorphisms of CYP3A5 and CYP3AP1 on cyclosporine dose requirement and concentration-to-dose ratio in renal allograft recipients. Cyclosporine-dependent renal allograft recipients were genotyped for CYP3A5 A6986G and CYP3AP1 G-44A. The cyclosporine dosages prescribed and the corresponding cyclosporine trough levels for each patient were recorded so that cyclosporine dose per weight (mg/kg/day) and concentration-to-dose ratio (C(0)/D, whereby C(0) is trough level and D is daily dose per weight) could be calculated. A total of 67 patients were recruited for our study. The dose requirement for 1, 3, and 6 months post-transplantation ranged 2.3-11.4, 1.0-9.0, and 1.4-7.2 mg/kg/day, respectively. Patients with *1*1*1*1 (n=5) CYP3A5- and CYP3AP1-linked genotypes needed higher dose of cyclosporine compared to patients with *1*3*1*3 (n = 27) and *3*3*3*3 (n = 33) linked genotypes in months 3 and 6 post-transplantation (P < 0.016). The identification of patients with *1*1*1*1 by CYP3A5 and CYP3AP1 genotyping may have a clinically significant and positive impact on patient outcome with reduced rejection rate by providing pretransplant pharmacogenetic information for optimization of cyclosporine A dosing.
Angiotensin II type 1 receptor (AGTR1) has been reported to play a fibrogenic role in non-alcoholic fatty liver disease (NAFLD). In this study, five variants of the AGTR1 gene (rs3772622, rs3772627, rs3772630, rs3772633, and rs2276736) were examined for their association with susceptibility to NAFLD. Subjects made up of 144 biopsy-proven NAFLD patients and 198 controls were genotyped using TaqMan assays. The liver biopsy specimens were histologically graded and scored according to the method of Brunt. Single locus analysis in pooled subjects revealed no association between each of the five variants with susceptibility to NAFLD. In the Indian ethnic group, the rs2276736, rs3772630 and rs3772627 appear to be protective against NAFLD (p = 0.010, p = 0.016 and p = 0.026, respectively). Haplotype ACGCA is shown to be protective against NAFLD for the Indian ethnic subgroup (p = 0.03). Gene-gene interaction between the AGTR1 gene and the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, which we previously reported as associated with NAFLD in this sample, showed a strong interaction between AGTR1 (rs3772627), AGTRI (rs3772630) and PNPLA3 (rs738409) polymorphisms on NAFLD susceptibility (p = 0.007). Further analysis of the NAFLD patients revealed that the G allele of the AGTR1 rs3772622 is associated with increased fibrosis score (p = 0.003). This is the first study that replicates an association between AGTR1 polymorphism and NAFLD, with further details in histological features of NAFLD. There is lack of evidence to suggest an association between any of the five variants of the AGTR1 gene and NAFLD in the Malays and Chinese. In the Indians, the rs2276736, rs3772630 and rs3772627 appear to protect against NAFLD. We report novel findings of an association between the G allele of the rs3772622 with occurrence of fibrosis and of the gene-gene interaction between AGTR1gene and the much-studied PNPLA3 gene.