Coptotermes gestroi (Wasmann) or the Asian subterranean termite is a serious structural pest in urban settlements in Southeast Asia that has been introduced to other parts of the world through human commerce. Although mitochondrial DNA markers were previously used to shed light on the dispersal history of the Asian subterranean termite, there were limited attempts to analyze or include populations of the termite found in the wild in Southeast Asia. In this study, we analyzed the 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase subunit 1 (cox1) genes of Asian subterranean termite colonies found in mangrove swamps, beach forests, plantations, and buildings in semi-urban and urban areas to determine the relationship between colonies found in the wild and the urban habitat, and to investigate the possibility of different ecotypes of the termite in Peninsular Malaysia. Our findings show that the 16S rRNA haplotypes recovered from this study clustered into eastern, western, and southern populations of the termite, while the cox1 haplotypes were often specific to an area or site. The 16S rRNA and cox1 genes or haplotypes showed that the most abundant haplotype occupied a wide range of environments or habitats. In addition, the cox1 tree showed evidence of historical biogeography where basal haplotypes inhabited a wide range of habitats, while apical haplotypes were restricted to mangrove swamps and beach forests. Information on the haplotype-habitat association of C. gestroi will enable the prediction of habitats that may harbor or be at risk of invasion in areas where they have been introduced.
Rubella has been listed as a mandatory notifiable disease in Taiwan since 1988. Because of high coverage rates with an effective vaccine, rubella cases have decreased dramatically in Taiwan since 1994. However, rubella outbreaks still occur due to imported transmission. Five large clusters were detected in Taiwan from 2007 to 2011. In 2007, one cluster was caused by rubella genotype 1E viruses that were imported from Vietnam, whereas another cluster was caused by genotype 2B viruses and was untraceable. In 2008, two clusters were caused by different lineages of genotype 1E viruses that were imported from Malaysia. In 2009, a cluster that was caused by genotype 2B viruses was associated with imported cases from Vietnam. The rubella viruses from 124 confirmed cases from 2005 to 2011 were characterized, and the data revealed that these viruses were distributed in the following four genotypes: 1E (n = 56), 1h (n = 1), 1j (n = 4), and 2B (n = 63). Of these viruses, 93 (75%) were associated with imported cases, and 43 of 56 genotype 1E viruses were associated with imported cases from China, Vietnam, Malaysia, and Indonesia. One genotype 1h virus was imported from Belarus, and three of four genotype 1j viruses were imported from the Philippines. Of 63 rubella genotype 2B viruses, 46 were imported from Vietnam, Thailand, Malaysia, China, Germany, and South Africa. Molecular surveillance allows for the differentiation of circulating rubella viruses and can be used to investigate transmission pathways, which are important to identify the interruption of endemic virus transmission.
In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype B' that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region.
Three new species and one new variety of bioluminescent Mycena collected from Peninsular Malaysia are described herein. All new species belong to Mycena sect. Calodontes in what is known as the Mycena pura complex. Comprehensive descriptions, photographs, illustrations and comparisons with phenetically similar species are provided. Molecular sequences data from the nuclear internal transcribed spacers (ITS-1 and ITS-2, including the 5.8S rRNA) were used to infer relationships within sect. Calodontes. Axenic cultures were obtained to provide data on culture morphology. This is the first published photographic documentation of bioluminescent basidiomes of members of Mycena sect. Calodontes. Also, this addition brings the total known bioluminescent fungi to 77 species.
Mycena illuminans Henn. is described and re-evaluated based on recently collected material from peninsular Malaysia, providing comprehensive descriptions, illustrations and photographs. In addition to morphological data, axenic monokaryon and dikaryon cultures were established to provide data on culture morphology and the mating system of the species. Molecular sequences data from the nuclear large subunit (LSU) gene also are presented, confirming that M. illuminans is not a synonym of Mycena chlorophos.
Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5(') untranslated region (UTR) of hPPARalpha gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5(')-untranslated region of human PPARalpha gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARalpha4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment -341/+34, but lower in the region -341/-1147 as compared to the fragment -341/+34, indicating the presence of an element conferring transcriptional activation between positions -341 and +34 or alternatively, the presence of transcriptional repression between positions -341 and -1147 in the promoter B of hPPARalpha.
To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action.
A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide-virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive beta-turns around T2-L3-T4-T5 and K6-L7-Y8-C9. The other conformer exhibited a beta-hairpin bend type of structure with a bend around L3-T4-T5-K6.
Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.
Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTPS, gene symbol: PTS) is involved in the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), which is a vital cofactor of nitric oxide synthases and three types of aromatic amino acid hydroxylases; the latter are important enzymes in the production of neurotransmitters. We conducted a study of PTS mutations in East Asia, including Taiwan, Mainland China, Japan, South Korea, the Philippines, Thailand and Malaysia. A total of 43 mutations were identified, comprising 22 previously reported mutations and 21 new discovered mutations. Among these, the c.155A>G, c.259C>T, c. 272A>G, c.286G>A and c.84-291A>G mutations were the most common PTS mutations in East Asia, while the c.58T>C and c.243G>A mutations were, respectively, specific to Filipinos and Japanese originating from Okinawa. Further studies demonstrated that each of the mutations listed above was in linkage disequilibrium to a specific allele of polymorphic microsatellite marker, D11S1347. These results suggest the presence of founder effects that have affected these frequent mutations in East Asia populations. In this context, D11S1347 should become one of the most reliable polymorphic markers for use in prenatal diagnosis among PTPS deficient families, especially where mutations are yet to be identified.
Two Gram-stain-negative, non-fermentative bacterial strains, designated 11-0202(T) and 11-0607, were isolated from soil in South Korea, and four others, LUH 13522, LUH 8638, LUH 10268 and LUH 10288, were isolated from a beet field in Germany, soil in the Netherlands, and sediment of integrated fish farms in Malaysia and Thailand, respectively. Based on 16S rRNA, rpoB and gyrB gene sequences, they are considered to represent a novel species of the genus Acinetobacter. Their 16S rRNA gene sequences showed greatest pairwise similarity to Acinetobacter beijerinckii NIPH 838(T) (97.9-98.4 %). They shared highest rpoB and gyrB gene sequence similarity with Acinetobacter johnsonii DSM 6963(T) and Acinetobacter bouvetii 4B02(T) (85.4-87.6 and 78.1-82.7 %, respectively). Strain 11-0202(T) displayed low DNA-DNA reassociation values (<40 %) with the most closely related species of the genus Acinetobacter. The six strains utilized azelate, 2,3-butanediol, ethanol and dl-lactate as sole carbon sources. Cellular fatty acid analyses showed similarities to profiles of related species of the genus Acinetobacter: summed feature 3 (C16 : 1ω7c, C16 : 1ω6c; 24.3-27.2 %), C18 : 1ω9c (19.9-22.1 %), C16 : 0 (15.2-22.0 %) and C12 : 0 (9.2-14.2 %). On the basis of the current findings, it is concluded that the six strains represent a novel species, for which the name Acinetobacter kookii sp. nov. is proposed. The type strain is 11-0202(T) ( = KCTC 32033(T) = JCM 18512(T)).
Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.
Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.
Twenty percent of genes that encode for hypothetical proteins from Klebsiella pneumoniae MGH78578 were identified, leading to KPN00728 and KPN00729 after bioinformatics analysis. Both open reading frames showed high sequence homology to Succinate dehydrogenase Chain C (SdhC) and D (SdhD) from Escherichia coli. Recently, KPN00729 was assigned as SdhD. KPN00728 thus remains of particular interest as no annotated genes from the complete genome sequence encode for SdhC. We discovered KPN00728 has a missing region with conserved residues important for ubiquinone (UQ) and heme group binding. Structure and function prediction of KPN00728 coupled with secondary structure analysis and transmembrane topology showed KPN00728 adopts SDH-(subunit C)-like structure. To further probe its functionality, UQ was docked on the built model (consisting KPN00728 and KPN00729) and formation of hydrogen bonds between UQ and Ser27, Arg31 (KPN00728) and Tyr84 (KPN00729) further reinforces and supports that KPN00728 is indeed SDH. This is the first report on the structural and function prediction of KPN00728 of K. pneumoniae MGH78578 as SdhC.
Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.
The discus fish (Symphysodon aequifasciata) is a cichlid demonstrating advanced mode of parental care towards fry. Both male and female fish utilized epidermal mucus secreted from specialized epidermal cells to feed developing fry. We utilized proteomics to compare protein profile from parental and nonparental fish. Gel analysis revealed a total of 35 spots that were up-regulated in parental mucus. In tandem, another 18 spots were uniquely expressed in parental mucus. MS analysis of these spots identified proteins such as fructose biphosphate aldolase, nucleoside diphosphate kinase, and heat shock proteins, which are essential to support energy provision, cell repair and proliferation, stress mediation, and defense mechanism in parental fish during parental-care period. Concurrently, the detection of several antioxidant-related proteins such as thioredoxin peroxidase and hemopexin suggests a need to overcome oxidative stress during hypermucosal production in parental-care behavior. A C-type lectin was also found to be uniquely expressed in parental mucus and could have important role in providing antimicrobial defense to both parental fish and fry. In summary, our study shows that discus mucus proteome undergoes changes in protein expression during parental-care period.
The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.
Persistent high-risk human papillomavirus (HPV) infection is known to play an important role in the genesis of cervical cancer. Since new screening and prevention strategies, namely improved HPV testing and HPV vaccination have been aggressively promoted recently, it is crucial to investigate the HPV distribution in Malaysia in order to maximize their cost-effectiveness. This study was therefore conducted to assess the HPV type distribution in the most populous region, the state of Selangor. A total of 200 cervical swab samples were collected in two health-screening campaigns, and also from women attending obstetrics and gynecology clinics in several hospitals in Selangor. DNA extraction was performed and HPV DNA was detected via nested PCR using MY09/MY11 as outer primers and GP5+/GP6+ as inner primers which target the L1 gene of the viral genome. The purified PCR products were subjected to automated DNA sequencing to determine the HPV genotype. Out of 180 β-globin positive samples, 84 (46.7%) were positive for HPV DNA. The most common HPV type found was high-risk oncogenic type 16 (40%), followed by HPV type 18 (3.3%), HPV 33 (1.7%), HPV 31 (0.6%), and low-risk HPV 87 (0.6%). Our study confirmed that nested PCR method is highly sensitive in detecting HPV DNA even in low risk patients. Since a relatively high prevalence rate of HPV infection was found in this population, prompt healthcare policy changes to bring about implementation of early HPV vaccination program is desirable to prevent a high incidence of cervical cancer.
Study site: Gynaecology and Obstetrics Clinics in Selangor (Hospital Kajang, Hospital Serdang, and the Britannia Women and Children Specialist Centre)