Escherichia coli is an important etiologic agent of lower respiratory tract infections (LRTI). Multidrug-resistant E. coli EC302/04 was isolated from a tracheal aspirate, and its genome sequence is expected to provide insights into antimicrobial resistance as well as adaptive and virulence mechanisms of E. coli involved in LRTI.
The whole-genome sequence of Mycobacterium bolletii M24, isolated from the bronchoalveolar lavage fluid of a Malaysian patient, is reported here. The circular chromosome of 5,507,730 bp helped to clarify the taxonomic position of this organism within the M. abscessus complex and revealed the presence of proteins potentially important for pathogenicity in a human host.
We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.
Mycobacterium abscessus is a species of rapidly growing nontuberculous mycobacteria that is frequently associated with opportunistic infections in humans. Here, we report the annotated genome sequence of M. abscessus strain M94, which showed an unusual cluster of tRNAs.
Enterococcus sp. strain C1 is a facultative anaerobe which was coisolated with Citrobacter sp. strain A1 from a sewage oxidation pond. Strain C1 could degrade azo dyes very efficiently via azo reduction and desulfonation in a microaerophilic environment. Here the draft genome sequence of Enterococcus sp. C1 is reported.
Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.
Novosphingobium sp. strain Rr 2-17 is an N-acyl homoserine lactone (AHL)-producing bacterium isolated from the crown gall tumor of a grapevine. To our knowledge, this is the first draft genome announcement of a plant-associated strain from the genus Novosphingobium.
The genome of Mycobacterium massiliense M172, isolated from a human sputum sample, was sequenced using Illumina GA IIX technology and found to contain 5,204,460 bp, including putative genes for virulence and antibiotic resistance as well as a 92-kb genomic region most likely to correspond to a mycobacteriophage.
Mycobacterium massiliense has recently been proposed as a member of Mycobacterium abscessus subsp. bolletii comb. nov. Strain M154, a clinical isolate from the bronchoalveolar lavage fluid of a Malaysian patient presenting with lower respiratory tract infection, was subjected to shotgun DNA sequencing with the Illumina sequencing technology to obtain whole-genome sequence data for comparison with other genetically related strains within the M. abscessus species complex.
The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.
Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks.
The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.
Helicobacter pylori infection results in diverse clinical conditions ranging from chronic gastritis and ulceration to gastric adenocarcinoma. Among the multiethnic population of Malaysia, Indians consistently have a higher H. pylori prevalence as compared with Chinese and Malays. Despite the high prevalence of H. pylori, Indians have a relatively low incidence of peptic ulcer disease and gastric cancer. In contrast, gastric cancer and peptic ulcer disease incidence is high in Chinese. H. pylori strains from Chinese strains predominantly belong to the hspEAsia subpopulation while Indian/Malay strains mainly belong to the hspIndia subpopulation. By comparing the genome of 27 Asian strains from different subpopulations, we identified six genes associated with risk of H. pylori-induced peptic ulcer disease and gastric cancer. This study serves as an important foundation for future studies aiming to understand the role of bacterial factors in H. pylori-induced gastro-duodenal diseases.
Previous studies showed that venoms of the monocled cobra, Naja kaouthia from Thailand and Malaysia are substantially different in their median lethal doses. The intraspecific venom variations of N. kaouthia, however, have not been fully elucidated. Here we investigated the venom proteomes of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V) through reverse-phase HPLC, SDS-PAGE and tandem mass spectrometry. The venom proteins comprise 13 toxin families, with three-finger toxins being the most abundant (63-77%) and the most varied (11-18 isoforms) among the three populations. NK-T has the highest content of neurotoxins (50%, predominantly long neurotoxins), followed by NK-V (29%, predominantly weak neurotoxins and some short neurotoxins), while NK-M has the least (18%, some weak neurotoxins but less short and long neurotoxins). On the other hand, cytotoxins constitute the main bulk of toxins in NK-M and NK-V venoms (up to 45% each), but less in NK-T venom (27%). The three venoms show different lethal potencies that generally reflect the proteomic findings. Despite the proteomic variations, the use of Thai monovalent and Neuro polyvalent antivenoms for N. kaouthia envenomation in the three regions is appropriate as the different venoms were neutralized by the antivenoms albeit at different degrees of effectiveness.
Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.
Cytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as "orphan" CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.
More than 90% of spinal muscular atrophy (SMA) patients show homozygous deletion of SMN1 (survival motor neuron 1). They retain SMN2, a highly homologous gene to SMN1, which may partially compensate for deletion of SMN1. Although the promoter sequences of these two genes are almost identical, a GCC insertion polymorphism has been identified at c.-320_-321 in the SMN1 promoter. We have also found this insertion polymorphism in an SMN2 promoter in an SMA patient (Patient A) who has SMA type 2/3.
The mechanisms of action of AN5-1 against Gram-negative and Gram-positive bacteria were investigated by evaluations of the intracellular content leakage and by microscopic observations of the treated cells. Escherichia coli and Staphylococcus aureus were used for this investigation. Measurements of DNA, RNA, proteins, and β-galactosidase were taken, and the results showed a significant increase in the cultivation media after treatment with AN5-1 compared with the untreated cells. The morphological changes of treated cells were shown using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The observations showed that AN5-1 acts against E. coli and against S. aureus in similar ways, by targeting the cell wall, causing disruptions; at a high concentration (80 AU/ml), these disruptions led to cell lysis. The 3D AFM imaging system showed that at a low concentration of 20 AU/ml, the effect of AN5-1 is restricted to pore formation only. Moreover, a separation between the cell wall and the cytoplasm was observed when Gram-negative bacteria were treated with a low concentration (20 AU/ml) of AN5-1.
Peptides are of great interest to be used as vaccine antigens due to their safety, ease of manufacturing and specificity in generating immune response. There have been massive discoveries of peptide antigens over the past decade. However, peptides alone are poorly immunogenic, which demand co-administration with strong adjuvant to enhance their immunogenicity. Recently, fibril-forming peptides such as Q11 and lipoamino acid-based carrier have been identified to induce substantial immune responses when covalently linked to peptide epitope. In this study, we have incorporated either Q11 or lipoamino acids to a peptide epitope (J14) derived from M protein of group A streptococcus to develop self-adjuvanting vaccines. J14, Q11 and lipoamino acids were also conjugated together in a single vaccine construct in an attempt to evaluate the synergy effect of combining multiple adjuvants. Physicochemical characterization demonstrated that the vaccine constructs folded differently and self-assembled into nanoparticles. Significantly, only vaccine constructs containing double copies of lipoamino acids (regardless in conjugation with Q11 or not) were capable to induce significant dendritic cells uptake and subsequent J14-specific antibody responses in non-sizes dependent manners. Q11 had minimal impact in enhancing the immunogenicity of J14 even when it was used in combination with lipoamino acids. These findings highlight the impact of lipoamino acids moiety as a promising immunostimulant carrier and its number of attachment to peptide epitope was found to have a profound effect on the vaccine immunogenicity.