Clonaggio, espressione e caratterizzazione della porzione solubile di Anaerobic induced protein A (sAniA) di Neisseria meningitidis

Neisseria meninigitidis è facilmente esposto allo stress nitrosoattivo durante la colonizzazione e/o durante la malattia, ma può continuare a sopravvivere in ambienti poveri di ossigeno attraverso la via di denitrificazione. E' , inoltre, in grado di contrastare l'effetto tossico dell' ossido nitrico per la presenza della nitrito reduttasi (codificata dal gene aniA) primo anello del processo di denitrificazione. Questo studio ha, come scopo, quello di valutare la conservazione della sequenza nucleotidica e aminoacidica del gene aniA; l'espressione della proteina AniA di un gruppo di ceppi di N. meninigitidis isolati da casi di infezione meningococcica (invasivi) e da portatori (non invasivi). L' analisi della sequenza di 19 ceppi invasivi e in 4 ceppi non invasivi, ha mostrato un alto grado di somiglianza nonostante siano state trovate un certo numero di cambiamenti nucleotidici alcuni dei quali definiti codoni di stop ed altri responsabili di una perdita di attività dell' enzima. In particolare, in un ceppo invasivo (1332) è stata trovata una inserzione di 9 aminoacidi vicino al sito legante il rame di tipo I ed un cambiamento dell'istidina a leucina nel sito catalitico, in posizione 280. In due ceppi non invasivi è stata identificata una parte del gene codificante una trasposasi. Tutti i ceppi di meningococco rappresentanti le diverse alterazioni riconosciute nel gene aniA, esprimono, tuttavia, la proteina, eccetto in quei ceppi con una proteina incompleta. La crescita in vitro di ciascun ceppo di N. meningitidis è risultata essere paragonabile sia in presenza che in assenza di nitrito, ed il mancato consumo di nitrito, suggerisce che, nonostante la proteina venga espressa in tutti i ceppi, esiste probabilmente un difetto nel consumo di questo substrato da parte dellâ enzima AniA. Sono state studiate le proprietà biochimiche del dominio solubile dell' enzima AniA (sAniA) del ceppo di riferimento MC58, ottenuto clonando la sequenza del gene corrispondente nel vettore di espressione batterica pET28. In particolare, l' analisi biochimica di base di sAniA ha dimostrato una chiara dipendenza dell' attività catalitica in un ambiente acido. I risultati ci suggeriscono che la presenza del gene aniA conservato e funzionale non è indispensabile per la sopravvivenza del meningococco, nonostante questo enzima sia incluso in una via metabolica importante nel ciclo di vita di questo patogeno.Neisseria meningitidis is likely to be exposed to nitrosative stress during natural colonization in the human host and disease in general. Furthermore, N. meningitidis can live in the absence of oxygen replacing the aerobic pathway with nitrite (NO2-) based metabolism involving as a first step the activity of nitrite reductase (encoded by aniA gene). During the life cycles inside the host, for instance during mucosal invasion or during sepsis, N. meningitidis may continue to survive by the denitrificant pathway. Expression of the these genes by N. meningitidis during colonization and different phases of disease plays a crucial role in bacterial survival and thus in infection. On the basis of this knowledge, the present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate, were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification. The results suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival

Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

<p>Abstract</p> <p>Background</p> <p>Several mutations have been described as responsible for rifampicin resistance in <it>Neisseria meningitidis</it>. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE) combined with mass spectrometry.</p> <p>Results</p> <p>In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible.</p> <p>Conclusions</p> <p>The analysis of differentially expressed proteins suggests that an intricate series of events occurs in <it>N. meningitidis </it>rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the widespread use of a particular drug. This seems the case of rifampicin resistant meningococci.</p

meningococcal c conjugate vaccine effectiveness before and during an outbreak of invasive meningococcal disease due to neisseria meningitidis serogroup c cc11 tuscany italy

Abstract Introduction In Tuscany, Italy, where a universal immunization program with monovalent meningococcal C conjugate vaccine (MCC) was introduced in 2005, an outbreak of invasive meningococcal disease (IMD) due to the hypervirulent strain of Neisseria meningitidis C/cc11 occurred in 2015–2016, leading to an immunization reactive campaign using either the tetravalent (ACWY) meningococcal conjugate or the MCC vaccine. During the outbreak, IMD serogroup C (MenC) cases were also reported among vaccinated individuals. This study aimed to characterize meningococcal C conjugate vaccines (MenC-vaccines) failures and to estimate their effectiveness since the introduction (2005–2016) and during the outbreak (2015–2016). Methods MenC cases and related vaccine-failures were drawn from the National Surveillance System of Invasive Bacterial Disease (IBD) for the period 2006–2016. A retrospective cohort-study, including the Tuscany' population of the birth-cohorts 1994–2014, was carried out. Based on annual reports of vaccination, person-years of MenC-vaccines exposed and unexposed individuals were calculated by calendar-year, birth-cohort, and local health unit. Adjusted (by birth-cohort, local health unit, and calendar-year) risk-ratios (ARR) of MenC invasive disease for vaccinated vs unvaccinated were estimated by the Poisson model. Vaccine-effectiveness (VE) was estimated as: VE = 1-ARR. Results In the period 2006–2016, 85 MenC-invasive disease cases were reported; 61 (71.8%) from 2015 to 2016. Twelve vaccine failures occurred, all of them during the outbreak. The time-interval from immunization to IMD onset was 20 days in one case, from 9 months to 3 years in six cases, and ≥7 years in five cases. VE was, 100% (95%CI not estimable, p = 0.03) before the outbreak (2006–2014) and 77% (95%CI 36–92, p  Conclusions In Tuscany, MenC-vaccine failures occurred exclusively during the 2015–2016 outbreak. Most of them occurred several years after vaccination. VE during the outbreak-period was rather high supporting an effective protection induced by MenC-vaccines

Characterisation of Neisseria meningitidis C strains causing two clusters in the north of Italy in 2007 and 2008.

Two clusters of invasive meningococcal disease in the north of Italy both due to serogroup C/ST-11 clonal complex are here described. The objective of the investigation was to analyse the phenotype and the genotype of meningococci involved in the two clusters which were of national relevance due to the fatal outcome of the majority of cases (six of the total of 10 cases). All the strains were C:2a:P1.5 ST-11/ET-37 clonal complex. Two pulsed field gel electrophoresis (PFGE) and variable number tandem repeats (VNTR) profiles were identified, one for each cluster. VNTRs were different from those detected in Italy for C/ST-11 strains isolated from sporadic cases in the same period. This laboratory surveillance report highlights the importance and the crucial role of molecular characterisation to confirm the relatedness among meningococci responsible for clusters of cases

Cluster of invasive Neisseria meningitidis infections on a cruise ship, Italy, October 2012.

We describe a cluster of four cases of invasive meningococcal disease that occurred on a cruise ship sailing along the Italian coast in October 2012. All four cases were hospitalised with severe illness and one of them died. This report illustrates the importance of rapid implementation of emergency control measures such as administration of prophylaxis to all crew members and passengers to prevent the spread of the disease in such a close environment

Twenty years of surveillance of Invasive Meningococcal Diseases in Puglia, Italy

The study aims to investigate the change in the burden of disease and the microbiological characteristics of Invasive Meningococcal Disease (IMD) in Puglia in comparison with overall incidence from 1994 through 2014

A Novel tDCS Sham Approach Based on Model-Driven Controlled Shunting

Abstract Background Transcranial direct current stimulation (tDCS), a non-invasive brain stimulation technique able to transiently modulate brain activity, is surging as one of the most promising therapeutic solutions in many neurological and psychiatric disorders. However, profound limitations exist in current placebo (sham) protocols that limit single- and double-blinding, especially in non-naive subjects. Objective /hypothesis: To ensure better blinding and strengthen reliability of tDCS studies and trials, we tested a new optimization algorithm aimed at creating an "active" sham tDCS condition (ActiSham hereafter) capable of inducing the same scalp sensations perceived during real stimulation, while preventing currents from reaching the cortex and cause changes in brain excitability. Methods A novel model-based multielectrode technique —optimizing the location and currents of a set of small electrodes placed on the scalp— was used to control the relative amount of current delivered transcranially in real and placebo multichannel tDCS conditions. The presence, intensity and localization of scalp sensations during tDCS was evaluated by means of a specifically designed questionnaire administered to the participants. We compared blinding ratings by directly addressing subjects' ability to discriminate across conditions for both traditional (Bifocal-tDCS and -Sham, using sponge electrodes) and our novel multifocal approach (both real Multifocal-tDCS and ActiSham). Changes in corticospinal excitability were monitored based on Motor Evoked Potentials (MEPs) recorded via concurrent Transcranial Magnetic Stimulation (TMS) and electromyography (EMG). Results Subjects perceived Multifocal-tDCS and ActiSham similarly in terms of both scalp sensations and their localization on the scalp, whereas traditional Bifocal stimulation was rated as more painful and annoying compared to its Sham counterpart. Additionally, differences in scalp localization were reported for active/sham Bifocal-tDCS. As for MEPs amplitude, a main effect of stimulation was found when comparing Bifocal-Sham and ActiSham (F(1,13)= 6.67, p=.023), with higher MEPs amplitudes after the application of Bifocal-Sham. Conclusions Compared to traditional Bifocal-tDCS, ActiSham offers better participants' blinding by inducing very similar scalp sensations to those of real Multifocal tDCS both in terms of intensity and localization, while not affecting corticospinal excitability

Frontiers of robotic endoscopic capsules: a review

Digestive diseases are a major burden for society and healthcare systems, and with an aging population, the importance of their effective management will become critical. Healthcare systems worldwide already struggle to insure quality and affordability of healthcare delivery and this will be a significant challenge in the midterm future. Wireless capsule endoscopy (WCE), introduced in 2000 by Given Imaging Ltd., is an example of disruptive technology and represents an attractive alternative to traditional diagnostic techniques. WCE overcomes conventional endoscopy enabling inspection of the digestive system without discomfort or the need for sedation. Thus, it has the advantage of encouraging patients to undergo gastrointestinal (GI) tract examinations and of facilitating mass screening programmes. With the integration of further capabilities based on microrobotics, e.g. active locomotion and embedded therapeutic modules, WCE could become the key-technology for GI diagnosis and treatment. This review presents a research update on WCE and describes the state-of-the-art of current endoscopic devices with a focus on research-oriented robotic capsule endoscopes enabled by microsystem technologies. The article also presents a visionary perspective on WCE potential for screening, diagnostic and therapeutic endoscopic procedures

Radiomics and Magnetic Resonance Imaging of Rectal Cancer: From Engineering to Clinical Practice

While cross-sectional imaging has seen continuous progress and plays an undiscussedpivotal role in the diagnostic management and treatment planning of patients with rectal cancer, alargely unmet need remains for improved staging accuracy, assessment of treatment response andprediction of individual patient outcome. Moreover, the increasing availability of target therapies hascalled for developing reliable diagnostic tools for identifying potential responders and optimizingoverall treatment strategy on a personalized basis. Radiomics has emerged as a promising, still fullyevolving research topic, which could harness the power of modern computer technology to generatequantitative information from imaging datasets based on advanced data-driven biomathematicalmodels, potentially providing an added value to conventional imaging for improved patient manage-ment. The present study aimed to illustrate the contribution that current radiomics methods appliedto magnetic resonance imaging can offer to managing patients with rectal cancer