Feasibility and Safety of Local Treatment with Recombinant Human Tissue Factor Pathway Inhibitor in a Rat Model of Streptococcus pneumoniae Pneumonia

Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagulant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was associated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI resulted in high levels of rh-TFPI in bronchoalveolar lavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology revealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.     

Intra-aortic balloon counterpulsation in acute myocardial infarction: old and emerging indications

Background

Recent evidence questions the role of intra-aortic balloon counterpulsation (IABP) in the treatment of acute myocardial infarction (AMI) complicated by cardiogenic shock (CS). An area of increasing interest is the use of IABP for persistent ischaemia (PI). We analysed the use of IABP in patients with AMI complicated by CS or PI.

Methods

From 2008 to 2010, a total of 4076 patients were admitted to our hospital for primary percutaneous coronary intervention (PCI) for AMI. Out of those, 239 patients received an IABP either because of CS or because of PI. Characteristics and outcome of those patients are investigated.

Results

The mean age of the study population was 64 ± 11 years; 75 % were male patients. Of the patients, 63 % had CS and 37 % had PI. Patients with CS had a 30-day mortality rate of 36 %; 1-year mortality was 41 %. Patients with PI had a 30-day mortality rate of 7 %; 1-year mortality was 11 %.

Conclusions

Mortality in patients admitted for primary PCI because of AMI complicated by CS is high despite IABP use. Outcome in patients treated with IABP for PI is favourable and mandates further prospective studies.     

Daily physical activity in ankylosing spondylitis: validity and reliability of the IPAQ and SQUASH and the relation with clinical assessments

Introduction

The aim of this study was to investigate the construct validity and test-retest reliability of the International Physical Activity Questionnaire (IPAQ; long form) and the Short QUestionnaire to Assess Health-enhancing physical activity (SQUASH) and to investigate the relation between daily physical activity and clinical assessments in patients with ankylosing spondylitis (AS).

Methods

For validity, the self-report questionnaires IPAQ and SQUASH were compared with daily physical activity assessed with the ActiGraph accelerometer during 7 consecutive days in 63 AS outpatients. For reliability, the IPAQ and SQUASH were administered twice approximately 1 week apart in 52 AS outpatients. In all 115 patients, clinical assessments were performed at the outpatient clinic.

Results

IPAQ and SQUASH total scores correlated significantly with accelerometer outcome: ρ = 0.38 and r = 0.35, respectively. Intraclass correlation coefficients between first and second assessments of the IPAQ and SQUASH were 0.83 and 0.89, respectively. Bland-Altman analyses showed no systemic bias, but in particular for the IPAQ the 95% limits of agreement were wide. Daily physical activity assessed by accelerometer, IPAQ, and SQUASH correlated significantly with disease activity, physical activity, and quality of life. A relation with spinal mobility was found only for the accelerometer and SQUASH. The direction of these correlations indicates that higher daily physical activity is related to lower disease activity and better physical function, spinal mobility and quality of life.

Conclusions

Both physical activity questionnaires showed modest construct validity. The SQUASH showed good test-retest reliability, superior to the IPAQ. These results indicate that the SQUASH is more suitable than the IPAQ to assess daily physical activity in AS population studies. However, it is desirable to add questions on AS-specific physical activity. Further studies are needed to investigate the causality of the relation between daily physical activity and clinical assessments.     

Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network

Background

Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity.

Methodology/Principal Findings

In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn₁₄) which displayed both a 125-fold increased capacity to inhibit KLK4 (K _i = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn₁₄ was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion.

Conclusion/Significance

These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.     

Simultaneous nutrient removal and lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW-04

The feasibility of using a microalga Chlorella vulgaris YSW-04 was investigated for removal of nutrients from piggery wastewater effluent. The consequent lipid production by the microalga was also identified and quantitatively determined. The wastewater effluent was diluted to different concentrations ranging from 20 to 80 % of the original using either synthetic media or distilled water. The dilution effect on both lipid production and nutrient removal was evaluated, and growth rate of C. vulgaris was also monitored. Dilution of the wastewater effluent improved microalgal growth, lipid productivity, and nutrient removal. The growth rate of C. vulgaris was increased with decreased concentration of piggery wastewater in the culture media regardless of the diluent type. Lipid production was relatively higher when using synthetic media than using distilled water for dilution of wastewater. The composition of fatty acids accumulated in microalgal biomass was dependent upon both dilution ratio and diluent type. The microalga grown on a 20 % concentration of wastewater effluent diluted with distilled water was more promising for generating high-efficient biodiesel compared to the other culture conditions. The highest removal of inorganic nutrients was also achieved at the same dilution condition. Our results revealed the optimal pretreatment condition for the biodegradation of piggery wastewater with microalgae for subsequent production of high-efficient biodiesel.     

Antisense RNA approach targeting Rep gene of Mungbean yellow mosaic India virus to develop resistance in soybean

The Yellow mosaic disease is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Yellow mosaic disease (YMD) is a major constraint to the production of soybean in South-East Asia. In India, yield losses of 10–88% had been reported due to YMD of soybean. An effort has been made to generate resistant soybean plants, by a construct targeting replication initiation protein (Rep) gene sequences of MYMIV. A construct containing the sequences of Rep gene (566?bp) in antisense orientation was used to transform cotyledonary node explants of three soybean cultivars (JS 335, JS 95-60 and NRC 37). Transformation efficiencies of 0.2, 0.21 and 0.24% were obtained with three soybean cultivars, JS 335, JS 95-60 and NRC 37, respectively. The presence of transgene in T₁ plants was confirmed by polymerase chain reaction (PCR) and sequence analysis. The level of resistance was observed by challenge inoculation with the virus in T₁ lines. The inheritance of transgene showed classical Mendelian pattern in six transgenic lines.     

Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases

UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.     

Streptococcus pneumoniae Serine Protease HtrA,but Not SFP or PrtA,Is a Major Virulence Factor in Pneumonia

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.     

Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3'-diindolylmethane contributes to inhibition of cell proliferation and induction of apoptosis in prostate cancer cells

Previous studies from our laboratory have shown anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane (DIM) through regulation of Akt and androgen receptor (AR) in prostate cancer cells. However, the mechanism by which DIM regulates Akt and AR signaling pathways has not been fully investigated. It has been known that FOXO3a and glycogen synthase kinase-3beta (GSK-3beta), two targets of activated Akt, interact with beta-catenin, regulating cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3beta, and beta-catenin are all AR coregulators and regulate the activity of AR, mediating the development and progression of prostate cancers. Here, we investigated the molecular effects of B-DIM, a formulated DIM with higher bioavailability, on Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling in hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We found that B-DIM significantly inhibited the phosphorylation of Akt and FOXO3a and increased the phosphorylation of beta-catenin, leading to the inhibition of cell growth and induction of apoptosis. We also found that B-DIM significantly inhibited beta-catenin nuclear translocation. By electrophoretic mobility shift and chromatin immunoprecipitation assays, we found that B-DIM inhibited FOXO3a binding to the promoter of AR and promoted FOXO3a binding to the p27(KIP1) promoter, resulting in the alteration of AR and p27(KIP1) expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive prostate cancer cells. These results suggest that B-DIM-induced cell growth inhibition and apoptosis induction are partly mediated through the regulation of Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling. Therefore, B-DIM could be a promising non-toxic agent for possible treatment of hormone-sensitive but most importantly hormone-refractory prostate cancers.     

Isolation of potentially pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx(1), stx(2), and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.