Expression levels of 10 candidate genes in lung tissue of vaccinated and TB‐infected cynomolgus macaques

The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomologus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1β, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non‐vaccinated animals.     

Soil hydrological properties as influenced by long‐term nitrogen application and landscape positions under switchgrass seeded to a marginal cropland

Switchgrass (Panicum virgatum L.) has the potential to recover the soil hydrological properties of marginal lands. Nitrogen (N) and landscape position are the key factors in impacting these soil properties under switchgrass. The specific objective of this study was to investigate the responses of N rate (low, 0 kg N/ha and high, 112 kg N/ha) and landscape positions (shoulder and footslope) on near‐surface soil hydrological properties that included: infiltration rate (q_s), saturated hydraulic conductivity (K_sat), bulk density (ρ_b), penetration resistance (SPR), water retention (SWR), pore‐size distribution (PSD), and carbon (C) and nitrogen (N) fractions under switchgrass production. Data showed that, in general, the N and landscape position significantly influenced soil hydrological properties. Higher N rate decreased ρ_b (1.23 and 1.36 g/cm³ at 0–5 and 5–15 cm, respectively) and SPR (1.06 and 1.53 MPa at 0–5 and 5–15 cm, respectively) at both depths and increased the q_s, K_sat and Green–Ampt estimated sorptivity (S) and hydraulic conductivity (K_s) parameters, and SWR (0–5 cm depth) at 0 and ?0.4 kPa matric potentials (ψ_m). Furthermore, footslope position significantly decreased ρ_b, SPR at 0–5 and 5–15 cm depths, and increased the q_s, K_sat, S, K_s, and SWR (0–5 cm depth) at every ψ_m ranged from 0 to ?30.0 kPa. The higher N rate increased the coarse mesopores (60–1,000 μm) and total pores, whereas, footslope position increased the coarse mesopores, micropores (<60 μm), and total pores. Data from this study showed that planting switchgrass with 112 kg N/ha under footslope position helped in improving the soil hydrological properties, those can be beneficial in enhancing the biomass yield under marginal lands.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Cohorting Dengue Patients Improves the Quality of Care and Clinical Outcome

IntroductionThe increasing incidence of dengue among adults in Malaysia and other countries has important implications for health services. Before 2004, in order to cope with the surge in adult dengue admissions, each of the six medical wards in a university hospital took turns daily to admit and manage patients with dengue. Despite regular in-house training, the implementation of the WHO 1997 dengue case management guidelines by the multiple medical teams was piecemeal and resulted in high variability of care. A restructuring of adult dengue inpatient service in 2004 resulted in all patients being admitted to one ward under the care of the infectious disease unit. Hospital and Intensive Care Unit admission criteria, discharge criteria and clinical laboratory testing were maintained unchanged throughout the study period.ObjectivesTo evaluate the impact of cohorting adult dengue patients on the quality of care and the clinical outcome in a university hospital in Malaysia.MethodsA pre (2003) and post-intervention (2005–6) retrospective study was undertaken.InterventionCohorting all dengue patients under the care of the Infectious Disease team in a designated ward in 2004.ResultsThe number of patients enrolled was 352 in 2003, 785 in 2005 and 1158 in 2006. The evaluation and detection of haemorrhage remained high (>90%) and unchanged throughout the study period. The evaluation of plasma leakage increased from 35.4% pre-intervention to 78.8% post-intervention (p = <0.001) while its detection increased from 11.4% to 41.6% (p = <0.001). Examination for peripheral perfusion was undertaken in only 13.1% of patients pre-intervention, with a significant increase post-intervention, 18.6% and 34.2% respectively, p = <0.001. Pre-intervention, more patients had hypotension (21.5%) than detected peripheral hypoperfusion (11.4%), indicating that clinicians recognised shock only when patients developed hypotension. In contrast, post-intervention, clinicians recognised peripheral hypoperfusion as an early sign of shock. The highest haematocrit was significantly higher post-intervention but the lowest total white cell counts and platelet counts remained unchanged. A significant and progressive reduction in the use of platelet transfusions occurred, from 21.7% pre-intervention to 14.6% in 2005 and 5.2% in 2006 post-intervention, p<0.001. Likewise, the use of plasma transfusion decreased significantly from 6.1% pre-intervention to 4.0% and 1.6% in the post-intervention years of 2005 and 2006 respectively, p<0.001. The duration of intravenous fluid therapy decreased from 3 days pre-intervention to 2.5 days (p<0.001) post-intervention; the length of hospital stay reduced from 4 days pre- to 3 days (p<0.001) post-intervention and the rate of intensive care admission from 5.8% pre to 2.6% and 2.5% post-intervention, p = 0.005.ConclusionCohorting adult dengue patients under a dedicated and trained team of doctors and nurses led to a substantial improvement in quality of care and clinical outcome.     

Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering

In this study, a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver particles (CS/nHAp/nAg) was developed by freeze drying technique, followed by introduction of silver ions in controlled amount through reduction phenomenon by functional groups of chitosan. The scaffolds were characterized using SEM, FT-IR, XRD, swelling, and biodegradation studies. The testing of the prepared scaffolds with Gram-positive and Gram-negative bacterial strains showed antibacterial activity. The scaffold materials were also found to be non-toxic to rat osteoprogenitor cells and human osteosarcoma cell line. Thus, these results suggested that CS/nHAp/nAg bio-composite scaffolds have the potential in controlling implant associated bacterial infection during reconstructive surgery of bone.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Tannase enzyme production by entrapped cells of Aspergillus niger FETL FT3 in submerged culture system

The ability of immobilized cell cultures of Aspergillus niger FETL FT3 to produce extracellular tannase was investigated. The production of enzyme was increased by entrapping the fungus in scouring mesh cubes compared to free cells. Using optimized parameters of six scouring mesh cubes and inoculum size of 1 × 10⁶ spores/mL, the tannase production of 3.98 U/mL was obtained from the immobilized cells compared to free cells (2.81 U/mL). It was about 41.64% increment. The immobilized cultures exhibited significant tannase production stability of two repeated runs.     

Evaluation of intra-host variants of the entire hepatitis B virus genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.     

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Background

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/Principal Findings

Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/Significance

These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.     

Production of lipase from Pseudomonas gessardii using blood tissue lipid and thereof for the hydrolysis of blood cholesterol and triglycerides and lysis of red blood cells

The study demonstrates the production of lipase (LIP) from Pseudomonas gessardii using blood tissue lipid as the substrate for the hydrolysis of blood cholesterol and triglycerides. The lipase was purified with the specific activity of 828 U/mg protein and the molecular weight of 56 kDa. The maximum lipase activity was observed at the pH 7.0 and the temperature 37 °C. The amino acid composition of purified lipase was determined by HPLC. The mesoporous activated carbon (MAC) was used for the immobilization of lipase for the repeated use of the enzyme catalyst. The K (m) value of immobilized lipase (MAC-LIP) and the free lipase (LIP) was 0.182 and 1.96 mM, respectively. The V (max) value of MAC-LIP and LIP was 1.33 and 1.26 mM/min, respectively. The MAC and MAC-LIP were characterized by scanning electron microscopy (SEM). The hydrolysis study showed 78 and 100% hydrolysis of triglycerides and cholesterol, respectively, for LIP and 84 and 100% hydrolysis of triglycerides and cholesterol, respectively, for MAC-LIP at the reaction time of 1 h. The effect of lipase on cell wall lysis was carried out on the RBCs of blood plasma. Interestingly, 99.9% lysis of RBCs was observed within 2 h. SEM images and phase contrast microscopy confirmed the lysis of RBCs. This work provides a potential biocatalyst for the hydrolysis of blood cholesterol and triglycerides.