HMGB‐1 induces c‐kit+ cell microvascular rolling and adhesion via both toll‐like receptor‐2 and toll‐like receptor‐4 of endothelial cells

High-mobility group box 1 (HMGB-1) is a strong chemo-attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB-1–mediated adhesion and rolling of c-kit⁺ cells and assess whether toll-like receptor-2 (TLR-2) and toll-like receptor-4 (TLR-4) of endothelial cells or c-kit⁺ cells are implicated in the activation of downstream migration signals to peripheral c-kit⁺ cells. Effects of HMGB-1 on the c-kit⁺ cells/endothelial interaction were evaluated by a cremaster muscle model in wild-type (WT), TLR-2 (−/−) and Tlr4 (LPS-del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real-time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB-1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c-kit⁺ cells was more than 13-fold higher than that of the control group (14.6 ± 5.1 cells/mm² in ‘WT’ versus 1.1 ± 1.0 cells/mm² in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR-2 (−/−) and Tlr4 (LPS-del) mice compared to WT mice (1.5 ± 1.4 cells/mm² in ‘TLR-2 (−/−)’ and 2.4 ± 1.4 cells/mm² in ‘Tlr4 (LPS-del)’ versus 14.6 ± 5.1 cells/mm² in ‘WT’, P < 0.05). TLR-2 (−/−) and Tlr4 (LPS-del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB-1 mediates c-kit⁺ cell recruitment via endothelial TLR-2 and TLR-4.     

Terminal sterilization of BisGMA-TEGDMA thermoset materials and their bioactive surfaces by supercritical CO2

The development of biomaterials endowed with bioactive features relies on a simultaneous insight into a proper terminal sterilization process. FDA recommendations on sterility of biomaterials are very strict: a sterility assurance level (SAL) of 10(-6) must be guaranteed for biomaterials to be used in human implants. In the present work, we have explored the potential of supercritical CO(2) (scCO(2)) in the presence of H(2)O(2) as a low-temperature sterilization process for thermoset materials and their bioactive surfaces. Different conditions allowing for terminal sterilization have been screened and a treatment time-amount of H(2)O(2) relationship proposed. The selected terminal sterilization conditions did not notably modify the mechanical properties of the thermoset nor of their fiber-reinforced composites. This was confirmed by μCT analyses performed prior to and after the treatment. On the contrary, terminal sterilization in the presence of H(2)O(2) induced a slight decrease in the surface hardness. The treatment of the thermoset material with scCO(2) led to a reduction in the residual unreacted monomers content, as determined by means of high performance liquid chromatography (HPLC) analyses. Finally, it was found that a thermoset coated with a polysaccharide layer containing silver nanoparticles maintained a very high antimicrobial efficacy even after the scCO(2)-based terminal sterilization.     

Rapid and Serial Quantification of Adhesion Forces of Yeast and Mammalian Cells

Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM). In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.     

Quinolone resistance in absence of selective pressure: the experience of a very remote community in the Amazon forest

Background

Quinolones are potent broad-spectrum bactericidal agents increasingly employed also in resource-limited countries. Resistance to quinolones is an increasing problem, known to be strongly associated with quinolone exposure. We report on the emergence of quinolone resistance in a very remote community in the Amazon forest, where quinolones have never been used and quinolone resistance was absent in 2002.

Methods

The community exhibited a considerable level of geographical isolation, limited contact with the exterior and minimal antibiotic use (not including quinolones). In December 2009, fecal carriage of antibiotic resistant Escherichia coli was investigated in 120 of the 140 inhabitants, and in 48 animals reared in the community. All fluoroquinolone-resistant isolates were genotyped and characterized for the mechanisms of plasmid- and chromosomal-mediated quinolone resistance.

Principal Findings

Despite the characteristics of the community remained substantially unchanged during the period 2002–2009, carriage of quinolone-resistant E. coli was found to be common in 2009 both in humans (45% nalidixic acid, 14% ciprofloxacin) and animals (54% nalidixic acid, 23% ciprofloxacin). Ciprofloxacin-resistant isolates of human and animal origin showed multidrug resistance phenotypes, a high level of genetic heterogeneity, and a combination of GyrA (Ser83Leu and Asp87Asn) and ParC (Ser80Ile) substitutions commonly observed in fluoroquinolone-resistant clinical isolates of E. coli.

Conclusions

Remoteness and absence of antibiotic selective pressure did not protect the community from the remarkable emergence of quinolone resistance in E. coli. Introduction of the resistant strains from antibiotic-exposed settings is the most likely source, while persistence and dissemination in the absence of quinolone exposure is likely mostly related with poor sanitation. Interventions aimed at reducing the spreading of resistant isolates (by improving sanitation and water/food safety) are urgently needed to preserve the efficacy of quinolones in resource-limited countries, as control strategies based only on antibiotic restriction policies are unlikely to succeed in those settings.     

Analysis of diagnostic pathways for colon cancer

Colon cancer is a pathology that benefits largely from an early cure. There are screening guidelines well assessed and overall accepted in several world nations. The question is if they are actually applied in the healthcare practice. The difficulty in the analysis of the application of diagnostic pathways is in the necessity to do a sort of time travel in the past: to chose a group of patients that have the same diagnosis, say colon cancer, and to be able to trace the pathways that led to the diagnosis. By exploiting the ability of data mining techniques to extract sequences from large masses of raw data, it has been possible to reconstruct the actual diagnostic services accessed with larger frequency by the patients and even the sequence of accessed services. The results show that there is a large majority of patients that followed pathways differing from the standard guidelines. The study describes how the database was built, the techniques adopted to extract and group together the data and concludes with an analysis of the diagnostic pathways.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

Application of the growth substrate pulsed feeding technique to a process of chloroform aerobic cometabolism in a continuous-flow sand-filled reactor

A process of chloroform (CF) aerobic cometabolic biodegradation by a butane-growing consortium was studied for 354 days in a 2-m continuous-flow sand-filled reactor. The study was aimed at (a) investigating the oxygen/substrate pulsed injection as a tool to control the clogging of the porous medium, to attain a wide bioreactive zone and to reduce substrate inhibition on CF cometabolism; (b) developing a reliable model of CF cometabolism in porous media. While the continuous supply of butane rapidly led to the clogging of the porous medium due to excessive biomass growth, the testing of six types of oxygen/substrate pulsed feeding led to the identification of a feeding schedule capable to prevent aquifer clogging and to lead to the development of a long bioreactive zone and to satisfactory CF degradation performances. The tested model of aerobic cometabolism allowed a suitable interpretation of the experimental data as long as the ratio of CF degraded to butane consumed was ≤0.27 mg_CF mg_butane^?1. A long-term 1-D simulation of the best-performing schedule of pulsed oxygen/substrate supply extended to a 30-m aquifer length resulted in a 20-m bioreactive zone and in a 96% CF removal.     

Influence of amplitude cancellation on the accuracy of determining the onset of muscle activity from the surface electromyogram

The purpose of the study was to quantify the influence of amplitude cancellation on the accuracy of detecting the onset of muscle activity based on an analysis of simulated surface electromyographic (EMG) signals. EMG activity of a generic lower limb muscle was simulated during the stance phase of human gait. Surface EMG signals were generated with and without amplitude cancellation by summing simulated motor unit potentials either before (cancellation EMG) or after (no-cancellation EMG) the potentials had been rectified. The two sets of EMG signals were compared at forces of 30% and 80% of maximum voluntary contraction (MVC) and with various low-pass filter cut-off frequencies. Onset time was determined both visually and by an algorithm that identified when the mean amplitude of the signal within a sliding window exceeded a specified standard deviation (SD) above the baseline mean. Onset error was greater for the no-cancellation conditions when determined automatically and by visual inspection. However, the differences in onset error between the two cancellation conditions appear to be clinically insignificant. Therefore, amplitude cancellation does not appear to limit the ability to detect the onset of muscle activity from the surface EMG.