Stromal upregulation of lateral epithelial adhesions: Gene expression analysis of signalling pathways in prostate epithelium

Background  

Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process.     

Sensitivity analysis of periprosthetic healing to cell migration, growth factor and post-operative gap using a mechanobiological model

A theoretical rationale, which could help in the investigation of mechanobiological factors affecting periprosthetic tissue healing, is still an open problem. We used a parametric sensitivity analysis to extend a theoretical model based on reactive transport and computational cell biology. The numerical experimentation involved the drill hole, the haptotactic and chemotactic migrations, and the initial concentration of an anabolic growth factor. Output measure was the mineral fraction in tissue surrounding a polymethymethacrylate (PMMA) canine implant (stable loaded implant, non-critical gap). Increasing growth factor concentration increased structural matrix synthesis. A cell adhesion gradient resulted in heterogeneous bone distribution and a growth factor gradient resulted in homogeneous bone distribution in the gap. This could explain the radial variation of bone density from the implant surface to the drill hole, indicating less secure fixation. This study helps to understand the relative importance of various host and clinical factors influencing bone distribution and resulting implant fixation.     

Increased interleukin (IL)-8 and decreased IL-17 production in chronic obstructive pulmonary disease (COPD) provoked by cigarette smoke

Recently, involvement of IL-17 in development of COPD has been noticed. Unlike IL-8, the role of IL-17 in COPD remains controversial. In order to further understand mechanisms in cigarette smoke (CS) induced COPD, we investigated IL-17 and IL-8 levels in different stages of COPD patients, and time courses of IL-17 and IL-8 release in CS induced COPD rats. A total of 73 elderly patients with COPD and 31 healthy volunteers were recruited in the study. IL-17 and IL-8 levels in the sputum and plasma were measured, and number of differential cells was counted. A newly developed CS induced rat COPD model was employed to study time courses of IL-17 and IL-8 release and nucleated cell accumulation. The results showed that IL-8 levels in the sputum of severe COPD patients were elevated by 16.5-fold, but IL-17 levels were reduced by 4.8-fold. While IL-8 correlated with neutrophils, IL-17 correlated with monocytes and lymphocytes. Similarly, level of IL-8 was increased, but IL-17 was decreased in the bronchoalveolar lavage fluid (BALF) of CS rats. Time course study showed that increased IL-8 production in the BALF initiated at 6 weeks, but decreased IL-17 production started at 10 weeks following CS exposure. In conclusion, increased IL-8 level in COPD patients appears mainly secreted from neutrophils and macrophages, whereas decreased IL-17 level seems resulted from reduced number of monocytes or damaged epithelial cells. Increased IL-8 (a proinflammatory cytokine) secretion and decreased IL-17 (a protective cytokine of airways) release can both contribute to development of COPD.     

The E-cadherin (CDH1) -160C>A polymorphism associated with gastric cancer among Asians but not Europeans

E-cadherin (CDH1) is a tumor suppressor gene involved in epithelial cell-cell interactions and plays important roles in the etiology of gastric cancer. Studies reporting conflicting results on the role of -160C>A polymorphism in the CDH1 promoter region on gastric cancer risk led us to perform a meta-analysis to investigate this relationship. Thirteen published case-control studies including 2509 gastric cancer cases and 3687 controls were identified. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association. Overall, individuals with the variant genotypes were not associated with a significant gastric cancer risk (AA vs. CC: OR?=?1.04, 95% CI: 0.74-1.48; CA vs. CC: 1.02, 0.85-1.21; AA/CA vs. CC: 1.03, 0.86-1.22; AA vs. CA/CC: 1.03, 0.74-1.43). However, in the stratified analysis by ethnicity, significantly decreased gastric cancer risk was found among Asians in dominant model (AA/CA vs. CC: 0.84, 0.72-0.99). Further, when stratified by clinicopathologic characteristics of gastric cancer, no statistically significant result was observed for any analysis. The results suggested that the CDH1 -160C>A polymorphism may contribute to susceptibility to gastric cancer among Asians. Additional well-designed large studies will be required to validate this association in different populations.     

Role of magnesium and a phagosomal P-type ATPase in intracellular bacterial killing

Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.     

HDX-analyzer: a novel package for statistical analysis of protein structure dynamics

BACKGROUND: HDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2. IMPLEMENTATION AND RESULTS: HDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student's t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme. CONCLUSION: Statistical analysis provides crucial evaluation of whether a protein region is significantly protected or unprotected during the HDX mass spectrometry studies. Although there are several other available software programs to process HDX experimental data, HDXanalyzer is the first software program to offer multiple statistical methods to evaluate the changes in protein structure dynamics based on HDX mass spectrometry analysis. Moreover, the statistical analysis can be carried out for both m/z value and deuterium incorporation rate. In addition, the software package can be used for the data generated from a wide range of mass spectrometry instruments.     

The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC

A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15 min (25 min) at 37 °C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353 nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC??) of GM6001 and inhibitor A for both TACE and MMP-9.     

Polyhedral oligomeric silsesquioxane (POSS) suppresses enzymatic degradation of PCL-based polyurethanes

In this Article, we studied the enzymatic hydrolytic biodegradation behavior of a novel multiblock thermoplastic polyurethane (TPU) system, which incorporates polyhedral oligomeric silsesquioxane (POSS) into linear biodegradable thermoplastic polyurethanes containing poly(ε-caproactone) (PCL) and polyethylene glycol (PEG) blocks. The biodegradation behavior of POSS-PCL-PEG TPUs was characterized by proton nuclear magnetic resonance spectroscopy ((1)H NMR), differential scanning calorimetry (DSC), tensile tests, scanning electron microscopy (SEM), and wavelength dispersive X-ray spectrometry (WDS) after enduring 22-day accelerated enzymatic hydrolytic degradation tests. POSS incorporation significantly suppressed in vitro enzymatic hydrolytic degradation of PCL-PEG-based multiblock TPUs by a surface passivation mechanism. WDS observations revealed that the covalently bonded POSS moieties developed a near-continuous and robust POSS-layer after initial degradation, which prevented ester bonds of PCL from enzymatic attack, thereby inhibiting further degradation. These striking results provide a new strategy to fabricate the polyester-based biostable thermoplastic polyurethanes (TPUs) of potential use in long-term surgical implants.