The field hockey coaching process across both Malaysia and India favours a traditional, coach-centred approach of mastering technical skills in terms of game play parameters, fitness, intensity, and load training, whereas a tactical- and player-centred pedagogical approach still takes a backseat. On the other hand, the Teaching Games for Understanding (TGfU) model offers tactical-cognitive instruction and is gaining international recognition for its ability to produce intelligent players via a problem-solving approach in game play. Therefore, the purpose of this quasi-experimental study was to investigate the effect of TGfU compared to skill mastery instruction, termed as Skill Drill Technical (SDT), among Malaysian and Indian elite junior hockey players in term of the game play attributes of adjust and cover in 5 vs. 5 small-sided game play and game play intensity via heart rate (HR) at different points of game play. A total of n = 60 players with an average age of 15 ± 1.03 was selected via simple random sampling from both countries involved in this study and assigned equally to groups, with 15 per group for TGfU and for SDT across Malaysia and India. Gathered data were analysed using the ANOVA and ANCOVA techniques. Findings indicated that there were no significant differences for adjust in 5 vs. 5 game play between TGfU and SDT across Malaysia and India after the intervention. For cover, there was significant improvement for Malaysian players using the TGfU model compared to SDT. In contrast, there was no significant difference between these two models among the Indian players after the intervention. There was significant difference between these two models in terms of warm-up HR across the two countries, and HR was higher via TGfU. For HR immediately after the 5 vs. 5 game play intervention and HR after three minutes' recovery, Indian players with TGfU recorded a higher and significant difference compared to SDT. However, findings indicated no significant difference between these two instruction types among Malaysians, although TGfU proved to have higher HR intensity. Therefore, these findings reiterated that TGfU is a useful approach for game play to enhance intensity and cardiac output. In conclusion, for TGfU to be more relevant to the coaching environment, future research should link game play and physiological parameters. TGfU should able to overcome the barriers of tradition and cultural background that may hinder its momentum.
Model based physical education curriculum of Teaching Games for Understanding (TGfU) is still at early stage of implementation in Malaysian schools whereby the technical or skill-led model continues to dominate the physical education curriculum. Implementing TGfU seems to be problematic and untested in this environment. Therefore, this study examined, the effects that a revised model of TGfU compared to Skill Drill Technical (SDT) a technical model had on learning movement skills in Badminton, including returning to base, decision making and skill execution whilst performing in a doubles game play and also explored teachers' perceptions of navigating between the two models. Participants aged 15.5 ± 1.0 years, N = 32, school Badminton players were randomly selected and assigned equally into groups of TGfU and SDT. Reflective data was gathered from two experienced physical education teachers who were involved in this study. Findings indicated for movement to the base in doubles game play indicated significant improvement, after intervention via TGfU. As for decision-making and skill execution in doubles game play, analysis revealed no significant difference after intervention. Findings from teachers reflection, indicated the importance of mini game play in both TGfU and SDT models, as the students enjoyed, and built up positive attitudes for both winning or losing in game situations. However, when negotiating the TGfU model, the teacher found it difficult at times to execute the pedagogical model, as students needed guidance to discuss aspects related to tactics. However, to keep this pedagogical model viable further research findings ought to be circulated among teachers in Malaysia and similar Southeast Asian counties.
Meat quality is always subject to consumer scrutiny when purchasing from retail markets on
mislabeling as fresh meat. Repeated cycles of ‘freeze-thaw’ degrade the quality of meat. Existing
studies have primarily embarked on physical, chemical and biochemical changes induced by
variable storage conditions. The authentication of fresh versus thawed meat quality can be
further explored with the data involving a series of biochemical pathways that were largely
well-studied in living muscle tissues. However, these pathways are less predictable in postslaughter
condition where muscle turns to meat. In addition, there is far less known about how
various management or environmental stimuli impact these pathways, either by substrate load
or altered cellular environment during storage. Though the rate of post-slaughter metabolism
is quite important in driving meat quality development, it is also fairly well established.
Alternatively, the biochemical mechanisms responsible for the cessation of postmortem
metabolism, or protracted carbohydrate metabolism are particularly puzzling. Likewise,
there is little information about the relationship between volatility profiles of biomolecules
with regards to functional groups, enzymatic activity, protein solubility and protein surface
properties in meat during storage. The studies of these changes could be used to distinguish
between fresh and thawed meat.
Amniotic fluid (AF) serves as an excellent alternative source of pluripotent stem cells, as they are not bound with ethical issues and the stem cells are more primitive than adult stem (AS) cells. Hence, they have higher potential. Here we aim to isolate and characterize pluripotent stem cells from mid-term and full-term pregnant rat amniotic fluid. The results demonstrate the evidence of heterogeneous population of cells in the amniotic fluid and some of the cells morphology shows similarity with ES cells.
Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.
The tropical pathogen Burkholderia pseudomallei requires long-term parenteral antimicrobial treatment to eradicate the pathogen from an infected patient. However, the development of antibiotic resistance is emerging as a threat to this form of treatment. To meet the need for alternative therapeutics, we proposed a screen of natural products for compounds that do not kill the pathogen, but in turn, abrogate bacterial virulence. We suggest that the use of molecules or compounds that are non-bactericidal (bacteriostatic) will reduce or abolish the development of resistance by the pathogen. In this study, we adopted the established Caenorhabditis elegans-B. pseudomallei infection model to screen a collection of natural products for any that are able to extend the survival of B. pseudomallei infected worms. Of the 42 natural products screened, only curcumin significantly improved worm survival following infection whilst not affecting bacterial growth. This suggested that curcumin promoted B. pseudomallei-infected worm survival independent of pathogen killing. To validate that the protective effect of curcumin was directed toward the pathogen, bacteria were treated with curcumin prior to infection. Worms fed with curcumin-treated bacteria survived with a significantly extended mean-time-to-death (p < 0.0001) compared to the untreated control. In in vitro assays, curcumin reduced the activity of known virulence factors (lipase and protease) and biofilm formation. To determine if other bacterial genes were also regulated in the presence of curcumin, a genome-wide transcriptome analysis was performed on curcumin-treated pathogen. A number of genes involved in iron acquisition and transport as well as genes encoding hypothetical proteins were induced in the presence of curcumin. Thus, we propose that curcumin may attenuate B. pseudomallei by modulating the expression of a number of bacterial proteins including lipase and protease as well as biofilm formation whilst concomitantly regulating iron transport and other proteins of unknown function.
Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.
Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation.
Burkholderia pseudomallei (B. pseudomallei) has been shown to persist intracellularly in patients with melioidosis, until reactivated by decreasing immunocompetence. We have previously demonstrated by transmission electron microscopy, the internalisation of B. pseudomallei by human macrophages and the occurrence of phagosome-lysosome fusion.
Oxidative stress can occur in sepsis and infection, when overproduction of free radicals is not countered by the host antioxidant system, leading to impairment of host cellular functions. Various disease states are accompanied by the accumulation of 15-F2t-IsoP in biological fluids. These isoprostanes are considered as markers of oxidative stress, and inflammation and inflammatory mediators.
Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis.
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease of the tropics with high clinical mortality rates. To date, no vaccines are approved for melioidosis and current treatment relies on antibiotics. Conversely, common misdiagnosis and high pathogenicity of Bp hamper efforts to fight melioidosis. This bacterium can be isolated from a wide range of niches such as waterlogged fields, stagnant water bodies, salt water bodies and from human and animal clinical specimens. Although extensive studies have been undertaken to elucidate pathogenesis mechanisms of Bp, little is known about how a harmless soil bacterium adapts to different environmental conditions, in particular, the shift to a human host to become a highly virulent pathogen. The bacterium has a large genome encoding an armory of factors that assist the pathogen in surviving under stressful conditions and assuming its role as a deadly intracellular pathogen. This review presents an overview of what is currently known about how the pathogen adapts to different environments. With in-depth understanding of Bp adaptation and survival, more effective therapies for melioidosis can be developed by targeting related genes or proteins that play a major role in the bacteria's survival.
The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.
Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.
The PreS domain of hepatitis B virus (HBV) is believed to be involved in virion assembly and attachment to a hepatocyte receptor during infection. In order to study the functions of this region, we fused it to the g3p protein of bacteriophage M13 that allows the fusion protein to be displayed at the tip of the filament. The fusion protein was detected by the anti-E tag antibody on a Western blot. The polypeptide in a soluble form was produced by transfecting a non-suppressor E. coli host cell with the recombinant phagemid. The soluble protein was detected in cytoplasm, in the periplasmic space and also in the medium. The functional display of the PreS domain would provide an alternative means to study its interactions with the nuleocapsid and hepatocytes.
We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.
A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV). A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure. An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus. Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV. Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV. These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.
Diabetes mellitus is a predisposing factor of melioidosis, contributing to higher mortality rates in diabetics infected with Burkholderia pseudomallei. To investigate how diabetes alters the inflammatory response, we established a streptozotocin (STZ) -induced diabetic murine acute-phase melioidosis model. Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42-hr course of infection but the hyperglycaemic environment did not increase the bacterial burden. However, after 24 hr, granulocyte counts increased in response to infection, whereas blood glucose concentrations decreased over the course of infection. A genome-wide expression analysis of the STZ-diabetic murine acute melioidosis liver identified ~1000 genes whose expression was altered in the STZ-diabetic mice. The STZ-diabetic host transcriptional response was compared with the normoglycaemic host transcriptional response recently reported by our group. The microarray data suggest that the presence of elevated glucose levels impairs the host innate immune system by delaying the identification and recognition of B. pseudomallei surface structures. Consequently, the host is unable to activate the appropriate innate immune response over time, which may explain the increased susceptibility to melioidosis in the STZ-diabetic host. Nevertheless, a general 'alarm signal' of infection as well as defence programmes are still triggered by the STZ-diabetic host, although only 24 hr after infection. In summary, this study demonstrates that in the face of a B. pseudomallei acute infection, poor glycaemic control impaired innate responses during the early stages of B. pseudomallei infection, contributing to the increased susceptibility of STZ-induced diabetics to this fatal disease.
Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.