In silico genotyping of blood group alleles using WGS data: a comparative study of the Orang Asli in Peninsular Malaysia.

Differences in the distribution of RBC antigens defining the blood group types among different populations have been well established. Fewer studies exist that have explored the blood group profiles of indigenous populations worldwide. With the availability of population-scale genomic datasets, we have explored the blood group profiles of theOrang Aslis, who are the indigenous population in Peninsular Malaysia and provide a systematic comparison of the same with major global population datasets. Variant call files fromwhole genome sequence data (hg19) of 114 Orang Asli were retrieved from The Orang Asli Genome Project. Systematic variant annotations were performed using ANNOVAR and only those variants mapping back to genes associated with 43 blood group systems and transcription factors KLF1 and GATA1 were filtered. Blood group-associated allele and phenotype frequencies were determined and were duly compared with other datasets including Singapore SequencingMalay Project, aboriginal western desert Australians and global population datasets including The 1000 Genomes Project and gnomAD. This study reports four alleles (rs12075, rs7683365, rs586178 and rs2298720) of DUFFY, MNS, RH and KIDD blood group systems which were significantly distinct between indigenous Orang Asli and cosmopolitanMalaysians. Eighteen alleles that belong to 14 blood group systems were found statistically distinct in comparison to global population datasets. Although not much significant differences were observed in phenotypes of most blood group systems, major insights were observed when comparing Orang Asli with aboriginal Australians and cosmopolitanMalaysians.This study serves as the first of its kind to utilize genomic data to interpret blood group antigen profiles of the OrangAsli population. In addition, a systematic comparison of blood group profiles with related populations was also analysed and documented.

Hepatoprotective and antioxidant activities of Dicranopteris linearis leaf extract against paracetamol-induced liver intoxication in rats.

Context: Dicranopteris linearis L. (Gleicheniaceae) leaves have been reported to exert hepatoprotective activity.Objective: The hepatoprotective and antioxidant effects of ethyl acetate partition of D. linearis (EADL) are investigated.Materials and methods: EADL was subjected to antioxidant and anti-inflammatory studies, and phytochemical analyses. In vivo study involved six groups (n = 6) of overnight fasted Sprague Dawley rats. The test solutions [10% DMSO (normal), 10% DMSO (negative), 200 mg/kg silymarin (positive) or EADL (50, 250 or 500 mg/kg)] were administered orally once daily for 7 consecutive days followed by oral vehicle (only for normal) or hepatotoxic induction using 3 g/kg paracetamol (PCM).Results: EADL exerted ≈ 90% radical scavenging effects based on the DPPH and superoxide anion radical scavenging assays, high antioxidant capacity in the oxygen radical absorbance capacity assay (≈ 555,000 units), high total phenolic content (≈ 350 mg GAE/100 g extract) (p < 0.05), but low anti-inflammatory effect. EADL also attenuated PCM-induced liver intoxication as indicated by reduced level of serum liver enzymes; increased activity of endogenous enzymatic antioxidant (superoxide dismutase - 8.3 vs. 4.0 U/g tissue; catalase - 119 vs. 52 U/g tissue) and; reduced level of lipid peroxidation marker (2.7 vs. 5.0 µM). Preliminary screening of EADL revealed the presence of saponins, tannins and flavonoids with further HPLC analysis demonstrating the presence of rutin and quercetin.Discussion and conclusion: EADL exerted hepatoprotective and antioxidant activities; thus, these data support the potential use of D. linearis as a new source for future hepatoprotective drug development.

Relative proteome quantification of alpha, beta, gamma and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia.

This is the first report of QQQ-mass spectrometric identification and quantification of the Hb subunits, alpha, beta, delta and gamma globin peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the Bio-Rad cation-exchange chromatography of haemoglobin. The objectives were to assess the relationship of the quantity of the free alpha, beta, delta and gamma globin chains with the phenotypic diversity of beta-thalassaemias (ß-thal). The results demonstrate that the pools of free globin chains in red blood cells were correlating with the severity of the disease in patients with different phenotypes of ß-thal. The mechanism and the regulation of synthesis of free globin chains pool in a normal individual and in patients with different ß-thal phenotypes could arise from several mechanisms which will require further investigation. The role of the free globin pool in patients with ß-thal for development of novel therapeutic approaches based on these potential targets requires further investigation. Pertinent biomarkers improves the diagnosis of the ß-thal, especially in low-income countries where they are most common and allows more effective therapeutic intervention leading to more successful therapeutic outcome.

Antinociceptive effect of semi-purified petroleum ether partition of Muntingia calabura leaves

ABSTRACT Muntingia calabura L., Muntingiaceae, is a medicinal plant for various pain-related diseases. The aims of the present study were to determine the antinociceptive profile and to elucidate the possible mechanisms of antinociception of petroleum ether partition obtained from crude methanol extract of M. calabura leaves using various animal models. The antinociceptive profile of petroleum ether fraction (given oral; 100, 250 and 500 mg/kg) was established using the in vivo chemicals (acetic acid-induced abdominal constriction and formalin-induced paw licking test) and thermal (hot plate test) models of nociception. The role of glutamate, TRPV1 receptor, bradykinin, protein kinase C, potassium channels, and various opioid and non-opioid receptors in modulating the partition's antinociceptive activity was also determined. The results obtained demonstrated that petroleum ether partition exerted significant (p < 0.05) antinociception in all the chemicals-, thermal-, capsaicin-, glutamate-, bradykinin, and phorbol 12-myristate 13-acetate (PMA)-induced nociception models. The antinociceptive activity was reversed following pretreatment with opioid antagonists (i.e. naloxone, β-funaltrexamine, naltrindole and nor-binaltorphimine), and the non-opioid receptor antagonists (i.e. pindolol (a β-adrenoceptor), haloperidol (a non-selective dopaminergic), atropine (a non-selective cholinergic receptor), caffeine (a non-selective adenosinergic receptor), and yohimbine (an α2-noradrenergic)). In addition, pretreatment with L-arginine (a nitric oxide (NO) donor), NG-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase (NOS)), methylene blue (MB; an inhibitor of cyclic-guanosine monophosphate (cGMP) pathway), or their combination failed to inhibit petroleum ether partition's antinociception. In conclusion, petroleum ether partition exerts antinociceptive activity at the peripheral and central levels via the modulation of, partly, the opioid (i.e. µ, κ and δ) and several non-opioids (i.e. β-adrenergic, dopaminergic, cholinergic, adenosinergic, and α2-noradrenergic) receptors, glutamatergic, TRPV1 receptors, PKC and K+ channels systems, but not L-arg/NO/cGMP pathway.

Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain.

Mycobacterium tuberculosis is an acid fast bacterial species in the family Mycobacteriaceae and is the causative agent of most cases of tuberculosis. Here, we report the genomic features of Mycobacterium tuberculosis isolated from the cerebrospinal fluid (CSF) of a patient diagnosed with both pulmonary and extrapulmonary tuberculosis (TB). The isolated strain was identified as Mycobacterium tuberculosis PR08 (MTB PR08). Genomic DNA of the MTB PR08 strain was extracted and subjected to whole genome sequencing using MiSeq (Illumina, CA,USA). The draft genome size of MTB PR08 strain is 4,292,364 bp with a G + C content of 65.2%. This strain was annotated to have 4723 genes and 48 RNAs. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010895.

The Implication of the Polymorphisms of COX-1, UGT1A6, and CYP2C9 among Cardiovascular Disease (CVD) Patients Treated with Aspirin.

PURPOSE:   Enzymes potentially responsible for the pharmacokinetic variations of aspirin include cyclooxygenase-1 (COX-1), UDP-glucuronosyltransferase (UGT1A6) and P450 (CYP) (CYP2C9). We therefore aimed to determine the types and frequencies of variants of COX-1 (A-842G), UGT1A6 (UGT1A6*2; A541G and UGT1A6*3; A522C) and CYP2C9 (CYP2C9*3; A1075C) in the three major ethnic groups in Malaysia. In addition, the role of these polymorphisms on aspirin-induced gastritis among the patients was investigated. METHODS: A total of 165 patients with cardiovascular disease who were treated with 75-150 mg daily dose of aspirin and 300 healthy volunteers were recruited. DNA was extracted from the blood samples and genotyped for COX-1 (A-842G), UGT1A6 (UGT1A6*2 and UGT1A6*3) and CYP2C9 (CYP2C9*3; A1075C) using allele specific polymerase chain reaction (AS-PCR). RESULTS: Variants UGT1A6*2,*3 and CYP2C9*3 were detected in relatively high percentage of 22.83%, 30.0% and 6.50%, respectively; while COX-1 (A-842G) was absent. The genotype frequencies for UGT1A6*2 and *3 were significantly different between Indians and Malays or Chinese. The level of bilirubin among patients with different genotypes of UGT1A6 was significantly different (p-value < 0.05). In addition, CYP2C9*3 was found to be associated with gastritis with an odd ratio of 6.8 (95 % Cl OR: 1.39 - 33.19; P = 0.033). CONCLUSION: Screening of patients with defective genetic variants of UGT1A6 and CYP2C9*3 helps in identifying patients at risk of aspirin induced gastritis. However, a randomised clinical study of bigger sample size would be needed before it is translated to clinical use.

Mechanisms of gastroprotection of methanol extract of Melastoma malabathricum leaves.

BACKGROUND: Melastoma malabathricum L. (Melastomaceae) is a small shrub with various medicinal uses. The present study was carried out to determine the gastroprotective mechanisms of methanol extract of M. malabathricum leaves (MEMM) in rats. METHODS: The extract's mechanisms of gastroprotection (50, 250, 500 mg/kg) were studied using the pylorus-ligation in rat model wherein volume, pH, free and total acidity of gastric juice, and gastric wall mucus content were determined. The involvement of endogenous nitric oxide (NO) and sulfhydryl (SH) compounds in the gastroprotective effect of MEMM were also measured. MEMM was subjected to the antioxidant, anti-inflammatory and phytochemical analysis and HPLC profiling. RESULTS: MEMM contained various phyto-constituents with quercitrin being identified as part of them. MEMM and quercitrin: i) significantly (p < 0.05) reduced the volume and acidity of gastric juice while increasing the pH and gastric wall mucus content.; ii) significantly (p < 0.05) increased the level of SOD, GTP and GTR while significantly (p < 0.05) reduced the level of CAT, MPO and TBARS activities.; iii) exerted gastroprotective activity when assessed using the ethanol-induced gastric ulcer assay, which was reversed by N(G)-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase) and N-ethylmaleimide (NEM; a sulfhydryl (SH) blocker). MEMM inhibited the lipoxygenase (LOX) and xanthine oxidase (XO) activities with the highest affinity for the former while quercitrin showed high affinity for XO activity. CONCLUSIONS: MEMM exhibited a gastroprotective activity due partly to the presence of quercitrin, its antioxidant and anti-inflammatory activities, and via the modulation of NO and SH groups.

Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA.

Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.

Mechanisms of α-mangostin-induced antinociception in a rodent model.

OBJECTIVE: Elucidate the antinociceptive mechanisms of α-mangostin isolated from Garcinia malaccensis Linn. METHODS: Male mice/rats (n = 6/group) were used in this between-group study. To determine α-mangostin's antinociceptive profile, animals were given α-mangostin orally (3, 30, or 100 mg/kg) 60 min before the start of the abdominal constriction or formalin tests. In the hot plate test, the noxious stimulus was applied before and 60, 90, 120, 150, 180, and 210 min after treatment with test solutions. Positive controls received 100 mg/kg acetylsalicylic acid (ASA; oral) or 5 mg/kg morphine (intraperitoneal injection) for the abdominal constriction and hot plate tests, respectively, and either ASA or morphine for the formalin test. Negative controls received vehicle only. To explore α-mangostin's mechanisms of action, we performed (i) the hot plate test with naloxone (5 mg/kg) pretreatment to verify involvement of opioid receptors; (ii) the abdominal constriction test with 20 mg/kg l-arginine, N(G)-nitro-l-arginine methyl esters (l-NAME), methylene blue (MB), l-arginine plus l-NAME, or l-arginine plus MB or 10 mg/kg glibenclamide pretreatment to verify involvement of the l-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) and K+-ATP pathways; and (iii) the paw-licking test using capsaicin (1.6 µg capsaicin/paw), glutamate (10 µmol glutamate/paw), or phorbol 12-myristate 13-acetate (PMA; 0.05 µg/paw) to verify involvement of vanilloid receptors, the glutamatergic system, and protein kinase C (PKC). RESULTS: α-mangostin significantly inhibited nociception (p < .05) in all models. Only naloxone, l-arginine, methylene blue, PMA, and glibenclamide affected α-mangostin antinociception significantly (p < .05). CONCLUSION: α-mangostin exhibits peripheral and central antinociception through modulation of opioid and vanilloid receptors, the glutamatergic system, and the l-arginine/NO/cGMP/PKC/K(+)-ATP pathways.

Effect of methanol extract of Dicranopteris linearis against carbon tetrachloride-induced acute liver injury in rats.

BACKGROUND: Dicranopteris linearis (family Gleicheniaceae) has been reported to possess anti-inflammatory and antioxidant activities but no attempt has been made to study its hepatoprotective potential. The aim of the present study was to determine the hepatoprotective effect of methanol extracts of D. linearis (MEDL) against carbon tetrachloride (CCl4)-induced acute liver injury in rats. METHODS: 6 groups (n = 6) of rats received oral test solutions: 10% dimethyl sulfoxide (DMSO), 200 mg/kg silymarin, or MEDL (50, 250, and 500 mg/kg), once daily for 7 consecutive days, followed by hepatotoxicity induction with CCl4. Blood and liver were collected for biochemical and microscopic analysis. The extract was also subjected to antioxidant studies (e.g. 2, 2-diphenyl-1-picrylhydrazyl (DPPH)- and superoxide anion-radical scavenging assays, oxygen radical absorbance capacity (ORAC) test and total phenolic content (TPC) determination), phytochemical screening and HPLC analysis. RESULTS: Pretreatment with MEDL and silymarin significantly (P < 0.05) reduced the serum levels of AST, ALT and ALP, which were increased significantly (P < 0.05) in DMSO-pretreated group following treatment with CCl4. Histological analysis of liver tissues in groups pretreated with MEDL and silymarin showed mild necrosis and inflammation of the hepatocytes compared to the DMSO-pretreated group (negative control group). The MEDL showed higher DPPH- and superoxide anion-radical scavenging activity as well as high TPC and ORAC values indicating high antioxidant activity. CONCLUSIONS: MEDL exerts hepatoprotective activity that could be partly contributed by its antioxidant activity and high phenolic content, and hence demands further investigation.

Antinociceptive activity of methanolic extract of Muntingia calabura leaves: further elucidation of the possible mechanisms.

BACKGROUND: Muntingia calabura (Elaecoparceae) is a medicinal plant traditionally used, particularly, by the Peruvian people to alleviate headache and cold, pain associated with gastric ulcers or to reduce the prostate gland swelling. Following the recent establishment of antinociceptive activity of M. calabura leaf, the present study was performed to further elucidate on the possible mechanisms of antinociception involved. METHODS: The methanol extract of M. calabura (MEMC) was prepared in the doses of 100, 250 and 500 mg/kg. The role of bradykinin, protein kinase C, pottasium channels, and various opioid and non-opioid receptors in modulating the extract's antinociceptive activity was determined using several antinociceptive assays. Results are presented as Mean ± standard error of mean (SEM). The one-way ANOVA test with Dunnett's multiple comparison was used to analyze and compare the data, with P < 0.05 as the limit of significance. RESULTS: The MEMC, at all doses, demonstrated a significant (p < 0.05) dose-dependent antinociceptive activity in both the bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced nociception. Pretreatment of the 500 mg/kg MEMC with 10 mg/kg glibenclamide (an ATP-sensitive K+ channel inhibitor), the antagonist of µ-, δ- and κ-opioid receptors (namely 10 mg/kg ß-funaltrexamine, 1 mg/kg naltrindole and 1 mg/kg nor-binaltorphimine), and the non-opioid receptor antagonists (namely 3 mg/kg caffeine (a non-selective adenosinergic receptor antagonist), 0.15 mg/kg yohimbine (an α2-noradrenergic antagonist), and 1 mg/kg pindolol (a ß-adrenoceptor antagonist)) significantly (p < 0.05) reversed the MEMC antinociception. However, 10 mg/kg atropine (a non-selective cholinergic receptor antagonist), 0.15 mg/kg prazosin (an α1-noradrenergic antagonist) and 20 mg/kg haloperidol (a non-selective dopaminergic antagonist) did not affect the extract's antinociception. The phytochemicals screening revealed the presence of saponins, flavonoids, tannins and triterpenes while the HPLC analysis showed the presence of flavonoid-based compounds. CONCLUSIONS: The antinociceptive activity of MEMC involved activation of the non-selective opioid (particularly the µ-, δ- and κ-opioid) and non-opioid (particularly adenosinergic, α2-noradrenergic, and ß-adrenergic) receptors, modulation of the ATP-sensitive K+ channel, and inhibition of bradikinin and protein kinase C actions. The discrepancies in MEMC antinociception could be due to the presence of various phytochemicals.

Antinociceptive activity of a synthetic oxopyrrolidine-based compound, ASH21374, and determination of its possible mechanisms.

This study was carried out to determine the antinociceptive activity of a novel synthetic oxopyrrolidine-based compound, (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374), and to elucidate the involvement of the opioid, vanilloid, glutamate, and nitric oxide - cyclic guanosine monophosphate (NO/cGMP) systems in modulating the observed antinociception. ASH21374, in the doses of 2, 10, and 100 mg/kg body mass, was administered orally to mice 60 mins prior to exposure to various antinociceptive assays. From the results obtained, ASH21374 exhibited significant (P < 0.05) antinociceptive activity in the abdominal constriction, hot-plate, and formalin tests that was comparable with 100 mg/kg acetylsalicylic acid or 5 mg/kg morphine, respectively. ASH21374 also attenuated capsaicin- and glutamate-induced paw licking. Pre-treatment with 5 mg/kg naloxone significantly (P < 0.05) inhibited the activity in all assays, while pretreatment with 10 mg/kg ß-funaltraxamine, 1 mg/kg naltrindole, or 1 mg/kg nor-binaltorphimine significantly (P < 0.05) reversed the activity in the abdominal constriction test. l-Arginine, N(G)-nitro-l-arginine methyl esters (l-NAME), methylene blue, and their combinations, failed to inhibit the ASH21374 antinociceptive activity. In conclusion, ASH21374 demonstrated antinociceptive activities on the peripheral and central nervous systems, mediated through the activation of opioid receptors, inhibition of the glutamatergic system, and attenuation of vanilloid-mediated nociceptive transmission. Further studies have been planned to determine the pharmacological potential of ASH21374.

Effects of the total alkaloidal extract of Murraya koenigii leaf on oxidative stress and cholinergic transmission in aged mice.

Alzheimer's disease (AD) is characterized by signs of major oxidative stress and the loss of cholinergic cells. The present study was designed to investigate the role of the total alkaloidal extract from Murraya koenigii (MKA) leaves on age related oxidative stress and the cholinergic pathway in aged mice. Ascorbic acid (100 mg/kg, p.o.) was used as a standard drug. The MKA improved the level of protective antioxidants such as glutathione peroxidase (GPx), reduced glutathione (GSH), glutathione reductase (GRD), superoxide dismutase (SOD) and catalase (CAT) in brain homogenate at higher doses (20 and 40 mg/kg, p.o.). Moreover, a dose dependent decline was noted in lipid peroxidation (LPO) and the nitric oxide assay (NO) at all doses of MKA (10, 20 and 40 mg/kg, p.o.). Interestingly, significant progress was noted with the supplementation of MKA by an improvement of the acetylcholine (ACh) levels and a reduction in the acetylcholinesterase (AChE) activity in aged mouse brain. In addition, a significant elevation of serum albumin (ALBU), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and total protein as well as a decline in creatinine, total cholesterol, urea nitrogen and glucose levels with MKA also ameliorated the hepatic and renal functions in normal ageing process. The results showed the possible utility of Murraya koenigii leaves in neuroprotection against neurodegenerative disorders such as Alzheimer's disease.