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Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP- N -acetylgalactosamine (UDP-GalNAc), UDP- N -acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·g cdw -1 , according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced., (© 2017 The Author(s).)

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological-chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.

Unlabelled: Smoking and depression are related. Bupropion, the first non-nicotinic drug that is an effective treatment in smoking cessation, is a tricyclic antidepressant that inhibits neuronal uptake of serotonin, dopamine and norepinephrine in the thalamic nuclei., Objective: To assess if certain personality factors (anxiety or depression) might predict the efficacy of bupropion for smoking cessation., Method: The study was carried out in two smoking cessation clinics in Madrid and Barcelona. Fifty patients (21 men) declaring the desire to quit smoking were enrolled. Their mean age was 43.6 years (SD 8.75). The patients were treated with 300 mg of bupropion per day for one month and expired CO was monitored for 6 months. Personality factors were assessed on a hospital anxiety and depression scale (HADS). We evaluated whether there was a significant difference in HADS scores for patients who were still not smoking after 6 months and those who had not managed to quit., Results: The 50 patients were smokers of a mean 39 packs per year (SD 17.82) and had mean scores of 7.4 (SD 4.15) for anxiety and 5.8 (SD 3.93) for depression. Four patients (8%) were unable to complete the study. After one month, 28% of the patients smoked, after 3 months 56% smoked and after 6 months 58% still smoked. The patients who smoked during the first month had higher depression scores than did the non-smokers (p = 0.03). After 3 and 6 months the patients who had managed to continue not smoking were those who had higher anxiety scores than did those who still smoked (p = 0.0052 at 3 months and p = 0.017 at 6 months)., Conclusion: Patients who responded better to treatment with bupropion after 6 months of follow-up were those with higher anxiety scores on the HADS. Depression levels influenced outcome only during the first month.

Therapy of patients with chronic respiratory failure is mainly directed at minimizing symptoms in order to improve, or at least to prevent a deterioration of, patients' well-being. Under such circumstances, the perceived effect of therapies on patients' well-being and daily life represents the most important subjective outcome of treatment. Therefore, there is a need to provide a global estimate of health in patients on long term oxygen therapy or overnight home mechanical ventilation. The Maugeri Foundation Respiratory Failure Questionnaire (MRF28) is the first health status ("quality of life") questionnaire specifically developed for use in CRF and its items were selected to be applicable to patients with both obstructive and restrictive diseases. The Quality of Life Evaluation and Survival Study (QuESS) is a multinational study with the aim of re-evaluating the natural history of chronic respiratory failure in about 300 patients. To the authors knowledge, the Quality of Life Evaluation and Survival Study is the first study to evaluate the natural history of chronic respiratory failure in such a large number of subjects and with a complete set of data. In fact, both pathophysiologic and health status assessments will be made. Moreover, by collecting data on mortality, disease exacerbations and hospitalization, it will also be possible to verify the predictive ability of health status versus pathophysiology in terms of mortality and healthcare utilization.

Smoking cessation is a first-line treatment for patients with bronchial and pulmonary diseases. Various strategies have been developed to help patients quit. Bupropion, a drug initially developed as an antidepressant, has recently been shown to have effects that increase the ability of a smoker to quit. This descriptive study, enrolling 86 patients who volunteered for a smoking cessation program, assesses the use of 300 mg of bupropion over a 4-week period combined with 12 weeks of nicotine patch application at doses that were lowered every 4 weeks. Abstinence was achieved by 69% of patients after 6 months of follow-up and no significant side effects were described. The percentage decreased to 58.6% after one year of follow-up. No significant differences were found between success in quitting in this study and either the number of prior attempts to quit or concomitant respiratory disease. We conclude that bupropion combined with transdermal nicotine is a good option to aid patients to achieve smoking cessation.

Quitting smoking is a first-line treatment for patients with bronchial diseases. Continued smoking worsens the clinical course of chronic broncho-pulmonary diseases and increases the number of exacerbations. Specialists commonly insist on the need to quit smoking. This study sought to determine whether a percentage of patients seen in a respiratory medicine clinic continued to smoke while denying doing so. One hundred twenty-five subjects were studied consecutively. At a regular visit they were first asked about smoking; later, without prior warning, exhaled carbon monoxide (CO) was measured by co-oximetry. If CO was over 10 ppm, the subject was considered to have been smoking. We defined a patient as a "liar" if he or she denied smoking but had a reading of CO in exhaled air over 10. Of the 125 cases studied, 21 (17%) smoked while denying doing so. Among men the percentage was 21%, and among ex-smokers, the figure was 27%. The highest value, 34%, was found among patients with chronic obstructive pulmonary disease (COPD). We conclude, therefore, that a substantial proportion of patients lies to their physicians. A third of COPD patients, who are particularly sensitive to the toxic effects of smoking, try to mislead their doctors.

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.