MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88-/- mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2-/-, TLR4-/-, and TLR2/4-/- mice were similar to those of C57BL/6 mice; however, TLR4-/- and TLR2/4-/- mice had impaired antifungal responses. The phenotype of infected IL-1R1-/- mice was similar to that of MyD88-/- mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1-/- mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.     

T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria

In invertebrates, cellular and humoral components are evolved to maintain their body immunity and integrity. Both these factors respond to different antigens such as microorganisms, vertebrate erythrocytes and foreign proteins. In this article, we report a study of a lectin (HSL) involved in immune response in the echinoderm, sea cucumber (Holothuria scabra). Correlative studies indicate that the expression of this defensive lectin is induced by bacterial challenge, wherein cell wall glycoconjugates of bacteria are involved in lectin induction. HSL showed strong broad spectrum antibacterial activity against both gram-positive and gram-negative bacteria. Under in vitro conditions, purified HSL mediate agglutination of the test bacteria, there by indicating a possible mode of action in physiological situation.     

Molecular characterization of novel variants of interferon-tau (IFNT) gene in Garole breed of sheep (Ovis aries)

The survivability of embryo, especially during the early embryonic life is dependent on the effective maternal recognition of pregnancy. Interferon-tau (IFNT), secreted from the elongating blastocyst, acts as the primary signal for maternal recognition of pregnancy in ruminant ungulates. IFNT has been studied extensively in many domesticated and wild ruminant species. In the present study, we have cloned and characterized the IFNT gene of Garole sheep, a popular Indian micro-sheep breed, which is known across the world for its high prolificacy and fecundity. The 588 bp sequences of two variants of IFNT gene described in this study are novel variants, compared to the variants reported previously in sheep. It exhibited more than 96% identity with other ovine IFNT variants and phylogenetically placed in a single clad containing the ovine, caprine and musk ox IFNT variants. The IFNT of Garole sheep demonstrated the highest identity with the genomic derived and highly expressed ovine IFNT variants.     

Discovery of a highly orally bioavailable c-5-[6-(4-Methanesulfonyloxyphenyl)hexyl]-2-methyl-1,3-dioxane-r-2-carboxylic acid as a potent hypoglycemic and hypolipidemic agent

A series of novel 1,3-dioxane-2-carboxylic acid derivatives containing alkyl chain tether and substituted phenyl group as a lipophilic tail have been prepared as agonists of PPARalpha and gamma. c-5-[6-(4-Methanesulfonyloxyphenyl)hexyl]-2-methyl-1,3-dioxane-r-2-carboxylic acid 13c exhibited potent hypoglycemic and lipid lowering activity with high oral bioavailability in animal models.     

Comparison of monofunctional and multifunctional monomers in phosphate binding molecularly imprinted polymers

In this study, molecularly imprinted polymers (MIPs) prepared using a multifunctional and a monofunctional monomer were compared with respect to their affinities, selectivities, and imprinting efficiencies for organophosphates. This is of interest because multifunctional monomers have higher affinities than traditional monofunctional monomers for their target analytes and thus should yield MIPs with higher affinities and selectivities. However, polymers containing multifunctional monomer may also have a higher number of unselective, non-templated binding sites. This increase in background binding sites could lead to a decrease in the magnitude of the imprinting effect and in the selectivity of the MIP. Therefore, phosphate selective imprinted and non-imprinted polymers (NIPs) were prepared using a novel multifunctional triurea monomer. The binding properties of these polymers were compared with polymers prepared using a monofunctional monourea monomer. The binding affinities and selectivities of the monomers, imprinted polymers, and NIPs were characterized by NMR titration, binding uptake studies, and binding isotherms. MIPs prepared with the triurea monomer showed higher binding affinity and selectivity for the diphenylphosphate anion in organic solvents than the MIPs prepared with the monofunctional monomer. Surprisingly, the binding properties of the NIPs revealed that the polymers prepared using the multifunctional and monofunctional monomers were very similar in affinity and selectivity. Thus, the multifunctional monomers increase not only the affinity of the MIP but also enhance the imprinting effect.     

Exterior exposure estimation using a one-compartment toxicokinetic model with blood sample measurements

Exposure assessment of individuals exposed to certain chemicals plays an important role in the analysis of occupational—as well as environmental-health problems. Biological monitoring, as an alternative to direct environmental measurements, may be applied to relate the exterior exposure with the amount of individual intake. In this paper, we estimate individuals’ (inhalation) exposure retrospectively from their blood concentrations via a simplified one-compartment toxicokinetic model. Considering stochastic variations to the toxicokinetic model, the solution to the resultant stochastic differential equation (SDE), together with measurement error, is transformed into a dynamic linear state-space model. The unknown model parameters and the mean inhalation concentration are then estimated via Markov Chain Monte Carlo (MCMC) simulations. The proposed method is used in the analysis of the styrene data (Wang et al. in Occup Environ Med 53:601–605, 1996) to backward estimate the inhalation concentration, assuming it is unknown. The data analysis showed that the internal stochastic variations, often ignored in toxicokinetic model analysis, outweighed in standard deviation almost twice that of the measurement error. Also, the simulation results showed that the method performed relatively well to the approach considering measurement error only.

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Contract/grant sponsor: National Science Council of Taiwan (NSC 93-2118-M-032-004); National Health Research Institutes of Taiwan (BS-096-PP-11).     

Reactive hyperemia in the human liver

We tested whether hepatic blood flow is altered following central hypovolemia caused by simulated orthostatic stress. After 30 min of supine rest, hemodynamic, plasma density, and indocyanine green (ICG) clearance responses were determined during and after release of a 15-min 40 mmHg lower body negative pressure (LBNP) stimulus. Plasma density shifts and the time course of plasma ICG concentration were used to assess intravascular volume and hepatic perfusion changes. Plasma volume decreased during LBNP (-10%) as did cardiac output (-15%), whereas heart rate (+14%) and peripheral resistance (+17%) increased, as expected. On the basis of ICG elimination, hepatic perfusion decreased from 1.67 +/- 0.32 (pre-LBNP control) to 1.29 +/- 0.26 l/min (-22%) during LBNP. Immediately after LBNP release, we found hepatic perfusion 25% above control levels (to 2.08 +/- 0.48 l/min, P = 0.0001). Hepatic vascular conductance after LBNP was also significantly higher than during pre-LBNP control (21.4 +/- 5.4 vs. 17.1 +/- 3.1 ml.min(-1).mmHg(-1), P < 0.0001). This indicates autoregulatory vasodilatation in response to relative ischemia during a stimulus that has cardiovascular effects similar to normal orthostasis. We present evidence for physiological post-LBNP reactive hyperemia in the human liver. Further studies are needed to quantify the intensity of this response in relation to stimulus duration and magnitude, and clarify its mechanism.     

Primate population decline in response to habitat loss: Borajan Reserve Forest of Assam, India

Systematic studies of the Borajan Reserve Forest in Assam, India, were conducted in 1995, 1997, and 1998. Initially this small (5 km²) forest was inhabited by substantial numbers of five species of diurnal primates and the forest was typical of Reserve Forests in northwest upper Assam. About two thirds of the forest had canopy cover of 20 – 50% or more. Civil unrest, political problems and a lack of resources for Forest Department personnel, however, resulted in rapid degradation of the area. After three years less than one third of the forest had more than 20% canopy cover; all primate populations had declined dramatically and the small percentage of juveniles in each species indicated that all were in imminent danger of local extinction. There was no evidence of hunting or trapping nor any large scale logging. Forest degradation was due primarily to small scale harvesting of forest products, selective cutting, and collection of firewood. Although only hand tools were employed, the forest inexorably declined in response to these steady pressures. Borajan may be an object lesson as to what can happen but it need not be the harbinger of Assam’s future.     

Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii β-glucosidase

β-glucosidase (EC 3.2.1.21; BG) cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345 μmol/min/mg on pNPGlc and a half-life of 1.13 h when assayed with pNPGlc at pH 7.1 and 70 °C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.

     

SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.