Features and applications of Ent35-MccV hybrid bacteriocin: current state and perspectives.

Bacteriocins are peptides of ribosomal synthesis that are active against bacteria related to the producing strain. They have been widely used in the food industry as biopreservatives. The generation of hybrid peptides by combining the genes that encode two different bacteriocins has made it possible to study the mechanisms of action of the bacteriocins that compose them and also develop new peptides with improved biotechnological applications. Hybrid bacteriocins may be obtained in several ways. In our laboratory, by combining enterocin CRL35 and microcin V (Ent35-MccV), we obtained a broad-spectrum peptide that is active against both Gram-positive and Gram-negative bacteria. Ent35-MccV is sensitive to the action of intestinal proteases and is heat resistant, which makes it a good candidate for use as a biopreservative. For this reason, the peptide was tested in skim milk and beef burgers as food models. We also obtained more potent variants of the hybrid by modifying the central amino acid of the hinge region that connects the two bacteriocins. This review also discusses future applications and perspectives regarding the Ent35-MccV and other hybrid peptides.Key Points• Ent35-MccV is a new broad-spectrum bacteriocin.• The mechanism of action of bacteriocins can be studied using hybrid peptides.• Genetic engineering allows obtaining improved bacteriocin derivatives.• Hybrid peptides can be used in the food, pharmaceutical, and veterinary applications.

Obtaining an Ent35-MccV derivative with mutated hinge region that exhibits increased activity against Listeria monocytogenes and Escherichia coli.

The present paper describes the generation of derivatives from the hybrid peptide called Ent35-MccV, active against Gram-positive and Gram-negative bacteria. This peptide has a triple glycine hinge region between enterocin CRL35 and microcin V. In order to obtain variants of Ent35-MccV with greater biotechnological potential, a saturation mutagenesis was carried out in the hinge region. As a result, we obtained a bank of E. coli strains expressing different mutated hybrid bacteriocins in the central position of the hinge region. From all these variants, we found that the one bearing a tyrosine in the central region of the hinge (Ent35-GYG-MccV) is 2-fold more active against E. coli and 4-fold more active against Listeria than the original peptide Ent35-MccV. This derivative was purified and characterized. The development and evaluation of alternative hinges for Ent35-MccV represents a step forward in the bioengineering of antimicrobial peptides. This approach fosters the rational design of peptides with enhanced antimicrobial activity.

Conservative Treatment of a Complicated Crown-root Fracture Using Adhesive Fragment Reattachment and Composite Resin Restoration: Two Year Follow-up.

Crown-root fracture is one of the most challenging fracture types in the dental traumatology literature. Traumatized anterior teeth require quick functional and esthetic repair. In the case of a complex crown fracture of the maxillary left central incisor, requiring endodontic treatment, a fiber-reinforced post was used to create a central support stump to restore the dental morphology. This report describes the clinical procedures involved in the treatment. After two years of follow-up, the clinical and radiographic findings demonstrated that the adopted clinical protocol was successful and yielded healthy periodontal tissues with no signs of periradicular pathology.

Non-parametric error distribution analysis from the laboratory calibration of various rainfall intensity gauges.

The analysis of counting and catching errors of both catching and non-catching types of rain intensity gauges was recently possible over a wide variety of measuring principles and instrument design solutions, based on the work performed during the recent Field Intercomparison of Rainfall Intensity Gauges promoted by World Meteorological Organization (WMO). The analysis reported here concerns the assessment of accuracy and precision of various types of instruments based on extensive calibration tests performed in the laboratory during the first phase of this WMO Intercomparison. The non-parametric analysis of relative errors allowed us to conclude that the accuracy of the investigated RI gauges is generally high, after assuming that it should be at least contained within the limits set forth by WMO in this respect. The measuring principle exploited by the instrument is generally not very decisive in obtaining such good results in the laboratory. Rather, the attention paid by the manufacturer to suitably accounting and correcting for systematic errors and time-constant related effects was demonstrated to be influential. The analysis of precision showed that the observed frequency distribution of relative errors around their mean value is not indicative of an underlying Gaussian population, being much more peaked in most cases than can be expected from samples extracted from a Gaussian distribution. The analysis of variance (one-way ANOVA), assuming the instrument model as the only potentially affecting factor, does not confirm the hypothesis of a single common underlying distribution for all instruments. Pair-wise multiple comparison analysis revealed cases in which significant differences could be observed.

Storm water infiltration in a monitored green roof for hydrologic restoration.

The objectives of this study are to provide detailed information about green roof performance in the Mediterranean climate (retained volume, peak flow reduction, runoff delay) and to identify a suitable modelling approach for describing the associated hydrologic response. Data collected during a 13-month monitoring campaign and a seasonal monitoring campaign (September-December 2008) at the green roof experimental site of the University of Genova (Italy) are presented together with results obtained in quantifying the green roof hydrologic performance. In order to examine the green roof hydrologic response, the SWMS_2D model, that solves the Richards' equation for two-dimensional saturated-unsaturated water flow, has been implemented. Modelling results confirm the suitability of the SWMS_2D model to properly describe the hydrologic response of the green roofs. The model adequately reproduces the hydrographs; furthermore, the predicted soil water content profile generally matches the observed values along a vertical profile where measurements are available.

Functional expression of a recombinant toxin - rPnTx2-6 - active in erectile function in rat.

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function.

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.

Gastrointestinal side effects in chronic opioid users: results from a population-based survey.

BACKGROUND: Gastrointestinal side effects are commonly associated with opioid treatment for pain. AIM: To understand gastrointestinal side effects associated with opioid treatment. METHODS: This study was a population-based survey of adults in the US who use opioids to manage pain unrelated to cancer. Participants were recruited from an existing Web-enabled panel and a supplemental panel of individuals who previously indicated an interest in participating in Web-based surveys. RESULTS: Overall, 2055 individuals participated in the main phase of the survey. Fifty-seven per cent of participants reported having had constipation that they associated with opioid treatment, and 49% reported constipation in the previous 4 weeks. Thirty-six per cent of participants reported new or worsening constipation in the previous 4 weeks. Thirty-three per cent of participants reported constipation as their most bothersome symptom associated with opioid treatment, 13% reported nausea, 11% abdominal pain and 10% gas. Seventy-three per cent of the participants who reported any GI symptoms did not change the dosage level or frequency of use of opioids because of adverse events, which may be explained by the fact that 72% of participants used over-the-counter laxatives and 12% a prescription laxative. CONCLUSION: Constipation is a frequent and significant event occurring with opioid use.

An investigation of wash-off controlling parameters at urban and commercial monitoring sites.

The relationship between the parameters of the wash-off function and the controlling hydrologic variables are investigated in this paper, assuming that the pollutant generation process basically depends on the watershed rainfall-runoff response characteristics. Data collected during an intense monitoring program carried out by the Department of Environmental Engineering of the University of Genova (Italy) within a residential area, an auto dismantler facility, a tourism terminal and a urban waste truck depot are used to this aim. The observed runoff events are classified into different TSS mass delivery processes and the occurrence of the first flush phenomenon is also investigated. The correlation between the mathematical parameters describing the exponential process and the hydrological parameters of the corresponding rainfall-runoff event is analysed: runoff parameters and in particular the maximum flow discharge over the time of concentration of the drainage network are proposed as the controlling factor for the total mass of pollutant that is made available for wash-off during each runoff event.

Quality of stormwater runoff from paved surfaces of two production sites.

In order to investigate stormwater pollutant loads associated with different anthropic activities and the related pollutant build-up and wash-off processes, two pilot sites have been equipped in the Liguria Region (Italy) for monitoring first flush water quality in a gas station and an auto dismantler facility. TSS, COD, HCtot and heavy metals in dissolved form (Zn, Pb, Cu, Ni, Cd, Cr) have been analyzed during the monitoring campaign (started in February 2004). Stormwater flow and quality data collected in both production sites confirm that EMC values are significantly higher than those observed in an urban site. In the auto dismantler site, the EMC values for TSS, COD and HC largely exceed the standard values (EC 91/271). Contrary to urban surface runoff, scarce correlation between TSS and COD concentrations is observed in runoff from both production sites. The occurrence and nature of the pollutant load connected to first flush flows is discussed by inspection of the M(V)-curves that are provided for all monitored water quality parameters. Significant first flush phenomenon is evidenced for TSS and HC, while such clear behavior doesn't emerge for heavy metals. Hydrologic and climatic characteristics (ADWP, rainfall intensity/depth) appear to scarcely affect the build-up and wash-off processes.

Tipping bucket mechanical errors and their influence on rainfall statistics and extremes.

Based on the error figures obtained after laboratory tests over a wide set of operational rain gauges from the network of the Liguria region, the bias introduced by systematic mechanical errors of tipping bucket rain gauges in the estimation of return periods and other statistics of rainfall extremes is quantified. An equivalent sample size is defined as a simple index that can be easily employed by practitioner engineers to measure the influence of systematic mechanical errors on common hydrological practice and the derived hydraulic engineering design. A few consequences of the presented results are discussed, with reference to data set reconstruction issues and the risk of introducing artificial climate trends in the observed rain records.

On the properties of stochastic intermittency in rainfall processes.

In this work we propose a mixed approach to deal with the modelling of rainfall events, based on the analysis of geometrical and statistical properties of rain intermittency in time, combined with the predictability power derived from the analysis of no-rain periods distribution and from the binary decomposition of the rain signal. Some recent hypotheses on the nature of rain intermittency are reviewed too. In particular, the internal intermittent structure of a high resolution pluviometric time series covering one decade and recorded at the tipping bucket station of the University of Genova is analysed, by separating the internal intermittency of rainfall events from the inter-arrival process through a simple geometrical filtering procedure. In this way it is possible to associate no-rain intervals with a probability distribution both in virtue of their position within the event and their percentage. From this analysis, an invariant probability distribution for the no-rain periods within the events is obtained at different aggregation levels and its satisfactory agreement with a typical extreme value distribution is shown.

Voice restoration after circumferential pharyngolaryngectomy with free jejunum repair.

Speech restoration after circumferential pharyngolaryngectomy with free jejunal repair for advanced tumors of the hypopharyngo-esophageal tract remains a difficult problem to solve. We report here the results of secondary voice restoration in six patients who received a Provox 2 type prosthesis and intensive speech therapy after circumferential pharyngolaryngectomy with free jejunum repair. No patient had operative or post-operative complications due to insertion of the prosthesis. No patient had to have the prosthesis removed during the follow-up (8 to 14 months). Analysis of some acoustic parameters of voice (fundamental frequency, waveform perturbations) and qualitative characteristics of speech (intelligibility, pleasantness and acceptability) demonstrated that all the patients were able to produce satisfactory speech after tracheojejunum puncture and speech therapy and were satisfied with their own ability to communicate. Our results are reassuring and we therefore advise that in patients undergoing free jejunum flap reconstruction of the hypopharyngo-esophageal tract voice restoration should be attempted by placing a voice prosthesis through a secondary tracheo-esophageal puncture and providing intensive speech training.

Health care, treatment patterns and cost of services for patients infected with chronic hepatitis C virus in a large insured New England population.

The purpose of this study was to describe the cost of health care and the patterns of treatment of young patients (under 65 years of age) identified in health insurance claims as having received services for chronic hepatitis C virus (HCV) infection. We screened computerized claims from a US indemnity and managed care organization for out-patient and in-patient diagnoses related to HCV. Treatment patterns and costs of services were evaluated in the following sites of care: in-patient care, emergency room, hospital out-patient care, ambulatory office care and pharmaceuticals. There were 191 patients with chronic HCV-related claims in this study population (25 per 100 000), during 1995-97. Medical services and pharmaceutical costs in total (US$ 7.1 million) constituted a considerable cost in patients with chronic HCV-related claims during 1995-97. A subset of 98 patients were prescribed interferon-alpha with substantial variability in treatment regimens. Claims data provides a unique opportunity to estimate dollars paid for treatment patterns and health services in a 'real world' insured population and contributes to the understanding of health services for chronic HCV.

Integrated treatment in locally advanced carcinoma of the oropharynx.

BACKGROUND AND OBJECTIVES: Oropharyngeal carcinoma tends to be aggressive and deeply infiltrative of nearby sites, with an high incidence of lymph node metastases. The last treatment decision generally depends on the stage of the lesion and the patient's general status. Oropharyngeal tumor is generally treated by integrated treatments. METHODS: We retrospectively studied 115 patients with locally advanced oropharyngeal tumors treated in our institution with combined therapies compare the results in two different groups of patients (surgery plus radiotherapy and chemotherapy plus radiotherapy). RESULTS: The 3-year overall survival rate in patients who underwent surgery plus radiotherapy was 82% and in those who underwent chemotherapy plus radiotherapy was 49%. CONCLUSION: The results suggest that surgery followed by radiotherapy seems to be the best treatment in the case of locally advanced oropharyngeal tumor.

The risk of serious cardiac arrhythmias among cisapride users in the United Kingdom and Canada.

PURPOSE: Serious, although rare, ventricular arrhythmias and deaths have been reported in patients taking cisapride monohydrate. Without quantification of the risk involved, it is impossible to develop rational therapeutic guidelines. SUBJECTS AND METHODS: Arrhythmic events (sudden deaths and other events compatible with serious ventricular arrhythmias) were sought among 36,743 patients prescribed cisapride in the United Kingdom and Saskatchewan, Canada. Prescriptions and cases were identified from computerized medical claims data and physicians' office records. We compared rates of events between periods of recent cisapride use and nonrecent use, using cohort analysis. Potential confounding factors, including concomitant treatment with agents that inhibit CYP3A4 metabolism or that prolong the QT interval, were assessed in a nested case-control study. RESULTS: In the cohort analysis, the incidence of the arrhythmic events was 1.6 times greater (95% confidence interval [CI]: 0.9 to 2.9) in periods of recent use. With adjustment for clinical history, use of CYP3A4 inhibitors, and use of drugs that prolong the QT interval, the odds ratio for cisapride and cardiac outcomes was 1.0 (95% CI: 0.3 to 3.7). There was no identifiable increase in risk when cisapride was dispensed at about the same time as QT-prolonging drugs or CYP3A4 inhibitors. QT-prolonging agents were associated with a 2.5-fold increase in the risk of arrhythmic events (95% CI: 1.1 to 5.8). CONCLUSIONS: Serious rhythm disorders were not associated with cisapride use, although the upper confidence bounds do not rule out an increase in risk.

Immune regulatory properties of corticosteroids: prednisone induces apoptosis of human T lymphocytes following the CD3 down-regulation.

Glucocorticoid hormones (GCH) induce apoptosis in PHA-primed peripheral blood T lymphocytes (PBL) and down-regulate membrane-bound proteins involved in the immune response. We have analyzed whether GCH are able to affect the expression of the TCR-associated molecules CD3, CD4, and CD8 on PBL-PHA, and whether the modulation of those receptors is related to the GCH-driven apoptosis of the PBL-PHA. Lymphocytes were cultured with PHA or with PHA plus prednisone (PDN) 10(-3), 10(-6), and 10(-9) M. Then expression of CD2, CD3, CD4, CD8, and CD56 antigens was studied by cytofluorimetric assay using propidium iodide (PI) staining and annexin procedure, and by gel electrophoresis of low molecular weight DNA. PDN, at a pharmacological concentration (10(-6) M), was able to inhibit the CD3 expression on T cells. The kinetics of CD3 decrement and of apoptosis show that the down-regulation of CD3 molecules precedes DNA fragmentation and that the cells lacking CD3 are those prone to PDN-induced apoptosis. The inhibition of CD3 is not related to a transcriptional or posttranscriptional phenomenon, because both PBL-PHA and PBL-PHA-PDN expressed the same amount of intracytoplasmic CD3 molecule. PDN also induced a down-regulation of the CD4 and CD8 molecules that resulted sooner in more intense CD8. In vitro PDN is able to induce apoptosis in PBL-PHA through a down-regulation of CD3 molecules.

Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds.

Different glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor.

IL-15/IL-15R alpha intracellular trafficking in human cells and protection from apoptosis.

IL-15 is an immunostimulatory cytokine sharing with IL-2 the IL-2R beta gamma complex. In vivo, IL-15 detection in synovial fluids has been associated with the development of rheumatoid arthritis. A debate exists as to whether IL-15 has the potential to be secreted in meaningful amounts or to act as a pericellular cytokine. Our data show (1) the presence of two IL-15 isoforms displaying signal peptides of different length and the capacity to be secreted restricted to the isoform bearing the longer one; (2) in cells expressing the two isoforms, the existence of different nuclear localization and intracellular trafficking of IL-15 and IL-15R alpha; and (3) an intercellular microcirculation of IL-15, not detectable with ELISA kits, but displaying a role as an anti-apoptotic factor able to induce the deflection of the TNFR associated factor 2 (TRAF) to IL-15R alpha. Our data point to a juxtacrine mechanism of action of IL-15 and suggest a role for IL-15/IL-15R alpha in the regulation of apoptosis.

Vascular pedicle rib graft in anterior transthoracic fusion procedures. Technical note.

A method is described in which anterior fusion of the thoracic vertebral column is performed using a rib strut graft maintained on its vascular pedicle. This straightforward technique is useful in selected patients undergoing anterior thoracic fusion procedures and can be used in conjunction with other anterior spinal implants. By maintaining bone graft blood supply, this technique promotes an optimum fusion environment, which may enhance the speed of graft incorporation and the ultimate strength of the construct.