Optimization of metallo-keratinase production by Pseudomonas sp. LM19 as a potential enzyme for feather waste conversion

Locally isolated bacterium Pseudomonas sp. LM19, a metallo-keratinase producer was used to hydrolyze the highly rigid keratin recalcitrant in this study. The production of crude keratinase by Pseudomonas sp. LM19 is influenced by both physical and nutritional parameters. The highest keratinase activity of 127?U/ml (2.15-fold) was observed in feather meal medium supplemented with fructose and peptone at a C/N ratio of 40. The optimum pH and temperature for keratinase production were found to be pH 8 and 30?°C, using 1% (w/v) feather as substrate. The degradation rate of the feathers was increased 2.4-fold at optimized physical and nutritional conditions. Feather degradation by Pseudomonas sp. LM19 led to the production of free amino acids such as arginine, glycine, leucine, and serine. The information on the production of keratinase by Pseudomonas sp. LM19 obtained from this study warrants further research for possible commercial application.     

Properties of STAT1 and IRF1 enhancers and the influence of SNPs

Background

STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.

Results

We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.

Conclusions

These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.

     

Metal complexes of a new class of polydentate Mannich bases: Synthesis and spectroscopic characterisation

A new class of polydentate Mannich bases featuring an N₂S₂ donor system, bis((2-mercapto-N-phenylacetamido)methyl)phosphinic acid H₃L¹ and bis((2-mercapto-N-propylacetamido)methyl)phosphinic acid H₃L², has been synthesised from condensation of phosphinic acid and paraformaldehyde with 2-mercaptophenylacetamide W1 and 2-mercaptopropylacetamide W2, respectively. Monomeric complexes of these ligands, of general formula K₂[Cr^III(Lⁿ)Cl₂], K₃[M′^II(Lⁿ)Cl₂] and K[M(Lⁿ)] (M′ = Mn(II) or Fe(II); M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II) or Hg(II); n = 1, 2) are reported. The structures of new ligands, mode of bonding and overall geometry of the complexes were determined through IR, UV–Vis, NMR, and mass spectral studies, magnetic moment measurements, elemental analysis, metal content, and conductance. These studies revealed octahedral geometries for the Cr(III), Mn(II) and Fe(II) complexes, square planar for Ni(II) and Cu(II) complexes and tetrahedral for the Co(II), Zn(II), Cd(II) and Hg(II) complexes. Complex formation studies via molar ratio in DMF solution were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1).     

Immunogenicity of Orally Administrated Recombinant <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang Expressing <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> Surface Adhesion Protein P23 in Mice

Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.     

PLTP deficiency improves the anti-inflammatory properties of HDL and reduces the ability of LDL to induce monocyte chemotactic activity

We reported that phospholipid transfer protein (PLTP) deficiency decreased atherosclerosis in mouse models. Because the decreased atherosclerosis was accompanied by a significant decrease in plasma HDL levels, we examined the properties of PLTP knockout (PLTP0) HDL and tested its ability to prevent LDL-induced monocyte chemotactic activity in human artery wall cell cocultures. We isolated HDL and LDL from LDL receptor knockout/PLTP knockout (LDLr0/PLTP0) mice and from apolipoprotein B transgenic (apoBTg)/PLTP0 mice as well as their controls. PLTP0 HDL was relatively rich in protein and depleted in phosphatidylcholine. Turnover studies revealed a 3.5- to 4.0-fold increase in the turnover of protein and cholesteryl ester in HDL from PLTP0 mice compared with control mice. The ability of HDL from LDLr0/PLTP0 and apoBTg/PLTP0 mice to prevent the induction of monocyte chemotactic activity in human artery wall cell cocultures exposed to human LDL was dramatically better than that in controls. Moreover, LDL from PLTP0 mice was markedly resistant to oxidation and induced significantly less monocyte chemotactic activity compared with that in controls. In vitro, PLTP0 HDL removed significantly more oxidized phospholipids from LDL than did control HDL. We conclude that PLTP deficiency improves the anti-inflammatory properties of HDL in mice and reduces the ability of LDL to induce monocyte chemotactic activity.     

Intracellular production of IFN-alpha 2b in Lactococcus lactis

Human interferon alpha (IFN-α) was expressed in two strains of Lactococcus lactis by aid of two promoters (P32 and Pnis) giving rise to two recombinant strains: MG:IFN and NZ:IFN, respectively. The expression of IFN was confirmed by ELISA and western blotting. Highest production was achieved using glucose for growth of both recombinant strains with nisin, used for induction of the recombinant strain with Pnis promoter, at 30 ng/ml. The optimum time for MG:IFN was 9 h and for NZ:IFN was 4.5 h. The highest productions by MG:IFN and NZ:IFN were 1.9 and 2.4 μg IFN/l, respectively. Both of the expressed IFNs showed bioactivities of 1.9 × 10⁶ IU/mg that were acceptable for further clinical studies.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

A hybrid of bees algorithm and flux balance analysis with OptKnock as a platform for in silico optimization of microbial strains

Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.     

Searching for a successful HDL-based treatment strategy

Despite strong evidence that HDL-cholesterol levels predict atherosclerotic events in a population, attempts at using an HDL-based treatment strategy have not yet been successful. Most of the efforts to date have focused on raising plasma HDL-cholesterol levels. This brief review focuses on a different strategy, which is based on the use of 18-amino acid apoA-I mimetic peptides. The story of these peptides spans decades and illustrates the remarkable complexity of HDL-based treatment strategies, but suggests that such a strategy may still be successful.     

Incidence and Characteristics of Injuries during the 2010 FELDA/FAM National Futsal League in Malaysia

Objective

In Malaysia, futsal is a popular sport played by individuals across all ages and genders. Despite its popularity, information on futsal related injury in Malaysia is not available. The purpose of this study is to examine the injury incidence and injury patterns among amateur men and women futsal players in Malaysia.

Methods

Players reported injury to the tournament medical team during the FELDA/FAM National Futsal League 2010 were interviewed and assessed by a Sports Medicine registrar. Player''s socio-demographic profiles and information about the injury were documented in the injury report form adapted from medical report form used by FIFA: Medical Assessment and Research Centre (F-Marc).

Results

A total of 86 injuries were reported from 141 matches, equivalent to an incidence of 91.5 injuries per 1000 player hours (95% CI 72.2 to 110.8), or 61.0 injuries per 1000 player matches (95% CI 48.1 to 73.9). Most were minor injuries resulted from contact with another player. Injuries often involved the lower extremity (44%) followed by the trunk (14%) and the upper limb (13%). Ankle (n = 7; 39%) and knee (n = 6; 33%) sprains were the most prevalent diagnoses of time-loss injuries. A significant association between time-loss and type of injury was found χ ² (1,N = 86) = 3.99, p = 0.04. In addition, time-loss injury was significantly associated with playing surface χ ² (1,N = 86) = 10.11, p = 0.018.

Conclusion

The injury rate during the FELDA/FAM National Amateur Futsal Men''s League in Malaysia was lower compared with previous Futsal World Cups competition. Most injuries resulted from contact with another player were minor and did not lead to time-loss from participation. Time-loss injury was significantly associated with type of injury and playing surface.