It is known that alpha1-antitrypsin deficiency is associated with emphysema in adults and liver cirrhosis in neonates. The phenotypes PiZZ and PiSZ are considered to be high risk groups. alpha1-antitrypsin deficiency is one of the most common lethal congenital disorders in Europe and the USA, occurring in approximately 1 in 2,000 caucasians of North European descent. Studies in Malaysia have found that the phenotypes PiZ and PiS are present in our population. Out of 950 samples analyzed, it was found that 10 samples were shown to be apparently Z homozygous phenotype. The phenotype is determined by high resolution isoelectrofocusing on an ultra-thin polyacrylamide gel embedded with narrow range Pi phamarlyte. The isoelectrofocused bands are confirmed by immunofixation and the plasma alpha1-antitrypsin levels determined by electroimmunoassay. The abnormal phenotypes are further confirmed by polymerase chain reaction using allele specific oligonucleotides.
The chromosome in situ suppression hybridization or chromosome painting technic was applied to confirm and eliminate the markers involving chromosome 21 segments using a chromosome 21 DNA library. The library ATCCLL21SNO2 was amplified, directly biotinylated using the polymerase chain reaction. The results demonstrated a translocation of chromosome 21 material on chromosome 2 and X and eliminate the origin of the marker. Thus, the technique provides an important tool to complement the conventional G-banding technic.
Matched MeSH terms: Abnormalities, Multiple/genetics*; Down Syndrome/genetics*; Intellectual Disability/genetics*
Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5'GGGCCC3'), SfiI (5'GGCCNNNNNGGCC3') and SmaI ('5CCCGGG3') enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.
Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.
Clinical studies were carried out on mild Indian sickle cell anaemia in Malaysia, and genetic and fertility studies were carried out on 101 families with and without sickle-cell haemoglobin (Hb S). The Indian sickle cell anaemia patients reached adulthood, and pregnancies and deliveries were uneventful without blood transfusion. There was no foetal wastage and the number of children produced was not significantly different from that in families without Hb S. 28 Indian patients hospitalized with Plasmodium falciparum malaria infection were also examined for their beta S genotype. P. falciparum malaria infection occurred much more frequently in individuals without Hb S than in Hb S carriers.
Talon cusps were observed in a 10-year-old boy and his 12-year-old sister. Although the sister's affected tooth was asymptomatic and free of any occlusal interference, the boy's tooth presented some problems. The affected tooth had displaced the occluding mandibular tooth lingually, and this had resulted in a mid-line shift. To overcome the problem, the talon cusp was ground off and a prophylactic odontotomy performed. Orthodontic treatment was later carried out.
The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.
Southeast Asian ovalocytosis (SAO) is a red blood cell abnormality common in malaria-endemic regions and caused by a 27 nt deletion of the band 3 protein gene. Since band 3 protein, also known as anion exchanger 1, is expressed in renal distal tubules, the incidence of SAO was examined in distal renal tubular acidosis (dRTA) in Malays in Kelantan, Malaysia. Twenty-two patients with dRTA and 50 healthy volunteers were examined for complication of SAO by both morphological and genetic analyses. SAO was identified in 18 of the 22 dRTA patients (81.8%), but only two of the 50 controls (4%). The incidence of SAO was significantly high in those with dRTA (p<0.001), indicating a dysfunctional role for band 3 protein/anion exchanger 1 in the development of dRTA.
Epstein-Barr virus (EBV) is believed to have a pathogenic role in lymphomas of the upper-aerodigestive tract. This study aims to elucidate the virus association pattern in nasal and nasal-type NK/T-cell lymphomas, and in sequential biopsies of these tumours. A total of 31 cases of previously diagnosed as lethal midline granuloma. Stewart's granuloma, nasal T-cell non-Hodgkin's lymphoma (T-NHL) and NK/T-cell lymphomas from all anatomical sites were retrieved from the files for the study. Reviews of these cases confirm 8 nasal T-NHL, 19 nasal and 4 extranasal lymphomas of NK/T-cell phenotype from 10 Malays, 18 Chinese, 2 Indian and 1 Kadazan. The male: female ratio was 2.4: 1. All T- and NK/T-cell lymphomas strongly expressed TIA-1 and 63% expressed CD2. The majority of NK/T-cell lymphoma occurred in Chinese (13/23), of which 12/13 (92%) of these cases were associated with EBV. Of the 15 nasal and 9 tonsillar B-cell lymphomas included for a comparison study, only 3 (20%) of the nasal cases were associated with EBV (1 male Chinese, 1 female Chinese and 1 male of other ethnic group). Eight cases of NK/T-cell tumours with sequential biopsies show persistence of EBV, irrespective of the interval and sites of subsequent presentations. This study confirms the cytotoxic nature of NK/T-cell tumour and that EBV is strongly associated with the disease regardless of the anatomical site of presentation and ethnicity. However, nasal and paranasal lymphomas of all phenotypes appear to show higher predilection of EBV association in the ethnic Chinese when compared to non-Chinese.
Matched MeSH terms: Herpesvirus 4, Human/genetics; Nose Neoplasms/genetics; Lymphoma, T-Cell/genetics
Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling.
The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.
Global trade increases plant introductions, but joint introduction of associated microbes is overlooked. We analyzed the ectomycorrhizal fungi of a Caribbean beach tree, seagrape (Coccoloba uvifera, Polygonacaeae), introduced pantropically to stabilize coastal soils and produce edible fruits. Seagrape displays a limited symbiont diversity in the Caribbean. In five regions of introduction (Brazil, Japan, Malaysia, Réunion and Senegal), molecular barcoding showed that seagrape mostly or exclusively associates with Scleroderma species (Basidiomycota) that were hitherto only known from Caribbean seagrape stands. An unknown Scleroderma species dominates in Brazil, Japan and Malaysia, while Scleroderma bermudense exclusively occurs in Réunion and Senegal. Population genetics analysis of S. bermudense did not detect any demographic bottleneck associated with a possible founder effect, but fungal populations from regions where seagrape is introduced are little differentiated from the Caribbean ones, separated by thousands of kilometers, consistently with relatively recent introduction. Moreover, dry seagrape fruits carry Scleroderma spores, probably because, when drying on beach sand, they aggregate spores from the spore bank accumulated by semi-hypogeous Scleroderma sporocarps. Aggregated spores inoculate seedlings, and their abundance may limit the founder effect after seagrape introduction. This rare pseudo-vertical transmission of mycorrhizal fungi likely contributed to efficient and repeated seagrape/Scleroderma co-introductions.
Survivin is a biomarker of cancer known for its anti-apoptotic and cell-cycle regulating properties. In the context of non-cancer pathology, high levels of survivin may be measured in blood and synovial fluid of patients with rheumatoid arthritis (RA) and associate with early joint damage and poor therapy response. The aim of the study was to investigate the value of survivin measurements in blood for diagnosis of RA in the frame of the Malaysian epidemiological investigation of rheumatoid arthritis (MyEIRA) study. The study enrolled RA patients from eight rheumatology centres in Peninsular Malaysia. The healthy controls matched by age, gender and ethnicity were recruited on the community basis from the residential area of the patients. Levels of survivin were measured in blood of RA patients (n = 1233) and controls (n = 1566) by an enzyme-linked immuno-sorbent assay (ELISA). The risk for RA was calculated as odds ratio (OR) and 95% confidence intervals in the individuals with high levels of survivin. The risk was calculated in relation to antibodies against cyclic citrullinated peptides (ACPA), detected by ELISA and HLA-DRB1 shared epitope (SE) alleles, identified by the polymerase chain reaction using sequence specific oligonucleotide method. High levels of survivin were detected in 625 of 1233 (50.7%) RA cases and in 85 of 1566 (5.4%) controls, indicating its high specificity for RA. Survivin was association with an increase in RA risk in the patients having neither SE-alleles nor ACPA (OR = 5.40, 95% CI 3.81-7.66). For the patients combining survivin, SE, and ACPA, the estimated risk for RA was 16-folds higher compared to the survivin negative patients with SE and ACPA(OR = 16.21, 95% CI 5.70-46.18). To conclude, detection of survivin in blood provides a simple test to improve diagnostic and to increase predictability for RA.
This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
Echinococcus granulosus is a worldwide zoonotic infection that causes human cystic echinococcosis (CE) or hydatid disease. The present study describes the isolation and production of a monoclonal antibody against recombinant AgB protein using the developed Human AntibodY Disease ENhanced (HAYDEN)-Filariasis library. The DNA sequences of the isolated clones were analyzed, followed by gene analysis and binding assays. Clone E1 showed a full-length sequence and represents the IgHV5-LV3 antibody gene family. The antibody protein yield was satisfactory, and it reacted specifically against rAgB. The novel E1 protein is potentially useful for the development of an antigen detection assay for CE. The ability of the Brugia malayi immune antibody library to isolate antibodies against Echinococcus granulosus antigens highlights the broad coverage of immune antibody libraries.
Thiocyanate (SCN-) forms as a by-product of cyanidation during gold ore processing and can be degraded by a variety of microorganisms utilizing it as an energy, nitrogen, sulphur and/or carbon source. In complex consortia inhabiting bioreactor systems, a range of metabolisms are sustained by SCN- degradation; however, despite the addition or presence of labile carbon sources in most bioreactor designs to date, autotrophic bacteria have been found to dominate key metabolic functions. In this study, we cultured an autotrophic SCN--degrading consortium directly from gold mine tailings. In a batch-mode bioreactor experiment, this consortium degraded 22 mM SCN-, accumulating ammonium (NH4+) and sulphate (SO42-) as the major end products. The consortium consisted of a diverse microbial community comprised of chemolithoautotrophic members, and despite the absence of an added organic carbon substrate, a significant population of heterotrophic bacteria. The role of eukaryotes in bioreactor systems is often poorly understood; however, we found their 18S rRNA genes to be most closely related to sequences from bacterivorous Amoebozoa. Through combined chemical and phylogenetic analyses, we were able to infer roles for key microbial consortium members during SCN- biodegradation. This study provides a basis for understanding the behaviour of a SCN- degrading bioreactor under autotrophic conditions, an anticipated approach to remediating SCN- at contemporary gold mines.
Alström syndrome (AS) is a monogenic syndromic ciliopathy caused by mutations in the ALMS1 (Alström Syndrome 1) gene. A total of 21 subjects with AS from 20 unrelated Chinese families were recruited. Our cohort consists of 9 females and 12 males, between 5 months and 20 years old. The first symptom(s) appeared between 3 and 24 months. They were recorded to be either visual impairments (83%) or dilated cardiomyopathy (17%). Median time from symptom onset to seeking medical attention was 6 months (3-36 months) and the median time needed to reach the final molecular diagnosis is 54 months (6-240 months). System involvement at the time of the survey was as follows: visual symptoms (100%), hearing Impairment (67%), endocrine symptoms (43%), neurological symptoms (19%), hepatic symptoms (14%), and renal Involvement (14%). These findings are comparable to data reported in the literature. However, the proportion of subjects with cognitive impairment (33%) and behavioral problems (19%) were higher. Thirty-three unique mutations were identified in the ALMS1 gene, of which 18 are novel mutations classified as pathogenic/likely pathogenic according to the American College of Medical Genetics (ACMG) guideline. Four recurrent mutations were identified in the cohort, in particular; c.2084C>A, p. (Ser695Ter), is suggestive to be a founder mutation in people of Chinese ancestry. The participation of AS subjects of differing ethnicities is essential to improve the algorithm in facial recognition/phenotyping, as well as to understand the mutation spectrum beyond than just those of European ancestry.
Oxidative stress has been linked to senescence and tumorigenesis via modulation of the cell cycle. Using a hydrogen peroxide (H2O2)-induced oxidative stress-induced premature senescence (OSIPS) model previously reported by our group, this study aimed to investigate the effects of oxidative stress on microRNA (miRNA) expression in relation to the G1-to-S-phase (G1/S) transition of the cell cycle and cell proliferation. On global miRNA analysis of the OSIPS cells, twelve significantly up- or down-regulated miRNAs were identified, the target genes of which are frequently associated with cancers. Four down-regulated miR-17 family miRNAs are predicted to target key pro- and anti-proliferative proteins of the p21/cyclin D-dependent kinase (CDK)/E2F1 pathway to modulate G1/S transition. Two miR-17 miRNAs, miR-20-5p and miR-106-5p, were confirmed to be rapidly and stably down-regulated under oxidative stress. While H2O2 treatment hampered G1/S transition and suppressed DNA synthesis, miR-20b-5p/miR-106a-5p over-expression rescued cells from growth arrest in promoting G1/S transition and DNA synthesis. Direct miR-20b-5p/miR-106a-5p regulation of p21, CCND1 and E2F1 was demonstrated by an inverse expression relationship in miRNA mimic-transfected cells. However, under oxidative stress, E2F1 expression was down-regulated, consistent with hampered G1/S transition and suppressed DNA synthesis and cell proliferation. To explain the observed E2F1 down-regulation under oxidative stress, a scheme is proposed which includes miR-20b-5p/miR-106a-5p-dependent regulation, miRNA-E2F1 autoregulatory feedback and E2F1 response to repair oxidative stress-induced DNA damages. The oxidative stress-modulated expression of miR-17 miRNAs and E2F1 may be used to develop strategies to retard or reverse MSC senescence in culture, or senescence in general.
Schistosoma malayensis n. sp., a member of the Schistosoma japonicum complex is described from Rattus muelleri in Peninsular Malaysia and 2 strains are characterized. The only morphological differences noted among adults from natural hosts were that S. malayensis are in general smaller than S. mekongi and S. japonicum. But these differences may be the result of host-induced variations and therefore are of little taxonomic value. To minimize the effects of host-induced variations, adult worms recovered from laboratory mice with similar worm burdens at 50-56 days postinfection were compared. These comparisons revealed only minor morphometric differences among these 3 species. Schistosoma malayensis eggs from naturally and experimentally infected hosts are most similar to those of S. mekongi, with eggs of both species being, in general, smaller than those of S. japonicum. The egg index for S. malayensis is usually higher than for S. japonicum and lower than for S. mekongi. Differences were noted in the developmental rates in mice for 2 isolates of S. malayensis, S. mekongi, and S. japonicum (Philippine strain), but relatively large differences observed between isolates of S. malayensis indicate that, in this case, the developmental rate is not a useful taxonomic character. Schistosoma malayensis is erected principally on the basis of differences, reported elsewhere, in the life histories and in the electrophoretic migration patterns of isoenzymes of adult worms as compared to S. mekongi and S. japonicum. These comparisons indicate that S. malayensis is more closely related to S. mekongi than to S. japonicum.
Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM). Here we have assessed the complexity of the OMP C-terminal β-barrel domains employed by H. pylori, and characterized the H. pyloriBAM complex. Around 50 Helicobacter-specific OMPs were assessed with predictive structural algorithms. The data suggest that H. pylori utilizes a unique β-barrel architecture that might constitute H. pylori-specific Type V secretions system. The structural and functional diversity in these proteins is encompassed by their extramembrane domains. Bioinformatic and biochemical characterization suggests that the low β-barrel-complexity requires only minimalist assembly machinery. The H. pylori proteins BamA and BamD associate to form a BAM complex, with features of BamA enabling an oligomerization that might represent a mechanism by which a minimalist BAM complex forms a larger, sophisticated machinery capable of servicing the outer membrane proteome of H. pylori.