Massive infection of Cystidicoloides ephemeridarum in brown trout Salmo trutta with skeletal deformities.

We investigated the cause of skeletal deformities found in brown trout from the Aspromonte mountain area in Reggio Calabria, Italy. Toxicological, histopathological and parasitological analyses were carried out on 14 fish with evident macro-morphological alterations from 2 different locations in the same river, and 4 control fish without morphological alterations from a different river (far from the first river but still within the area under study). Histopathological and radiological observations confirmed severe skeletal deformities in the specimens investigated. Parasitological examinations highlighted the presence of the nematode Cystidicoloides ephemeridarum, found only within the gastrointestinal tract of specimens showing deformities. Moreover, a direct correlation between parasite number and fish size was found. Given the low heavy metal levels and the presence of a massive parasitosis in teleosts showing deformities, we postulate a correlation between skeletal deformities and nematode infestation: the parasites caused a serious vitamin and mineral deficiency in the fish, which led to a dysplastic vertebral column. The low calcium levels found in malformed specimens compared with negative controls effectively confirm this hypothesis.

Gastric leiomyoma in a sea bass Dicentrarchus labrax broodfish.

This report describes a spontaneously arising non-infiltrative neoplasm of gastric tunica muscularis in a broodstock sea bass Dicentrarchus labrax from an Italian aquaculture farm. Microscopically, the mass was circumscribed and non-encapsulated and was composed of spindle cells arranged in parallel interlacing bundles or, occasionally, a whirling pattern. Cells had a small quantity of eosinophilic cytoplasm with distinct cell borders. Neoplastic cells were immuno-reactive with smooth muscle actin, vimentin and desmin; S100 was negative. The mucosal epithelium was intact with no neoplastic involvement. A gastric leiomyoma was diagnosed based on the findings. More efforts should be made to study the possible etiology of leiomyoma affecting fish from aquaculture.

An Enhanced GC/MS Procedure for the Identification of Proteins in Paint Microsamples.

The chemical characterization of materials used in works of art is extremely useful for gaining a better knowledge of the artistic heritage and to guarantee its preservation. A derivatization GC/MS procedure for the identification of proteins in a microsample from painted works of art has been optimized. The amino acid fraction is derivatized using anhydrous dimethylformamide (DMF) as solvent instead of pyridine (Py), commonly used to facilitate the reaction. Although pyridine is often considered a silylation catalyst, there are many instances in which silylation reactions actually are slower in pyridine than other solvents. In addition, pyridine also may have other undesirable effects such as the promotion of secondary products and other chromatographic anomalies. Using DMF, the formation of artifacts is limited and the derivatization yield of hydrophilic amino acids such as proline and hydroxyproline has improved, thus making the identification of organic paint media more straightforward. The method has been validated and successfully applied to identify the binder of the sample taken from the pictorial cycle of the 12th century monastery of Santa Maria delle Cerrate (Lecce, Italy), thus highlighting the use of eggs as a binding medium.

Seasonal variability of PM2.5 and PM10 composition and sources in an urban background site in Southern Italy.

Comparison of fine and coarse fractions in terms of sources and dynamics is scarce in southeast Mediterranean countries; differences are relevant because of the importance of natural sources like sea spray and Saharan dust advection, because most of the monitoring networks are limited to PM10. In this work, the main seasonal variabilities of sources and processes involving fine and coarse PM (particulate matter) were studied at the Environmental-Climate Observatory of Lecce (Southern Italy). Simultaneous PM2.5 and PM10 samples were collected between July 2013 and July 2014 and chemically analysed to determine concentrations of several species: OC (organic carbon) and EC (elemental carbon) via thermo-optical analysis, 9 major ions via IC, and 23 metals via ICP-MS. Data was processed through mass closure analysis and Positive Matrix Factorization (PMF) receptor model characterizing seasonal variabilities of nine sources contributions. Organic and inorganic secondary aerosol accounts for 43% of PM2.5 and 12% of PM2.5-10 with small seasonal changes. SIA (secondary inorganic aerosol) seasonal pattern is opposite to that of SOC (secondary organic carbon). SOC is larger during the cold period, sulphate (the major contributor to SIA) is larger during summer. Two forms of nitrate were identified: NaNO3, correlated with chloride depletion and aging of sea-spray, mainly present in PM2.5-10; NH4NO3 more abundant in PM2.5. Biomass burning is a relevant source with larger contribution during autumn and winter because of the influence of domestic heating, however, is not negligible in spring and summer, because of the contributions of fires and agricultural practices. Mass closure analysis and PMF results identify two soil sources: crustal associated to long range transport and carbonates associated to local resuspended dust. Both sources contributes to the coarse fraction and have different dynamics with crustal source contributing mainly in high winds from SE conditions and carbonates during high winds from North direction.

Facile synthesis of 3D flower-like Pt nanostructures on polypyrrole nanowire matrix for enhanced methanol oxidation.

Here we report the simple and rapid synthesis of three-dimension Pt flower-like nanostructures (PtNFs) on a polypyrrole nanowires (PPyNWs) matrix. Both PtNFs and PPyNWs are prepared by an electrochemical approach without using any seed, template or surfactant. The morphology and chemical composition of the resulting PtNF/PPyNWs hybrids are characterized by scanning electron microscopy and by X-ray photoelectron spectroscopy, respectively. Taking methanol oxidation as a model catalysis reaction, the electrocatalytic performance of the as-prepared PtNF/PPyNWs system has been evaluated by cyclic voltammetry and chronoamperometry, evidencing that these 3D materials exhibit excellent electrocatalytic activity and high level of poisoning tolerance to the carbonaceous oxidative intermediates. Such electrocatalytic performances can be ascribed to the combined effect of the flower-like structure promoting the exposure of more sites and the polymer nanowires matrix endorsing high dispersion of PtNF on a high electrochemically active surface area, besides the removal of sub-products from electrocatalytic sites.

Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella.

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS.

A self-administered questionnaire to screen the risk of dementia: a pilot study and the results of a comparison with the mini-mental state examination (MMSE).

Cognitive impairment is an age-related condition as the rate of cognitive decline rapidly increases with aging. The aim of this study was to screen the risk of cognitive decline in people over 60 years from 16 different Italian cities, by comparing the results of a self-administered questionnaire with the MMSE. We analyzed data from 203 persons aged 60 years and over, who voluntarily accepted to participate during the "Second Prevention Day for AD". A self-administered questionnaire, developed by clinicians of our Department of Aging, was distributed to all participants, in order to easily screen the risk of cognitive impairment. Then, all subjects underwent cognitive assessment by MMSE. We esteemed the risk of cognitive impairment of all participants basing on MMSE scores (no risk, mild and moderate risk) and we compared this assessment with the results obtained by the self-administered questionnaire. The comparison between the risk of cognitive impairment revealed by our questionnaire and the risk esteemed by MMSE resulted in a discrepancy in 43.96% of cases in no risk class. In mild risk group there was a discrepancy of results in 70.53% of subjects. In moderate risk class there was a discrepancy of results in 38.46% of individuals. Our questionnaire resulted to be accurate for the evaluation of patients with moderate risk of cognitive impairment. It showed a lower accuracy for the mild risk class, often overestimating the risk of cognitive decline.

Atrial fibrillation (AF) and cognitive impairment in the elderly: a case-control study.

AF is able not only to increase the risk of cognitive decline due to acute cerebrovascular events, but also to reduce cardiac output, with the consequence of impaired cerebral perfusion. The aim of this study was to evaluate the association between AF, dementia and depression in patients with negative anamnesis for past strokes. Our sample included 26 patients with a diagnosis of AF (paroxystic, persistent, permanent) and 31 patients with sinus rhythm, enrolled as controls. All selected patients underwent a Multidimensional Geriatric Assessment in order to investigate cognitive and behavioral functions. Statistical analysis of results showed a greater frequency of latent cognitive impairment in patients with AF, even in the absence of memory disorders. As a matter of facts, AF patients showed Mini Mental State Examination (MMSE) scores significantly lower than those with sinus rhythm (p<0.05) and Geriatric Depression Scale (GDS) scores higher than those without AF, evidencing a greater risk of depression too (p<0.02). Results showed a statistically significant association between AF, depression and cognitive impairment in early stage. In conclusion, AF is not only associated with the risk of developing cognitive impairment, but it can also be considered as a risk factor for dementia and depression, even in the absence of medical history of past stroke.

Role of cardiovascular risk factors (CRF) in the patients with mild cognitive impairment (MCI).

Few therapeutic options are available nowadays to improve the prognosis of patients with Alzheimer's disease (AD). There are rather several evidences in literature that controlling vascular risk factors may be an effective intervention for modifying the course of this disease. The aim of our study was to investigate the role of CRF in 50 patients with MCI according to Petersens's criteria, and to evaluate their influence on cognitive and behavioral features of the disease and on the development of dementia. Statistical analysis of the data showed that the 60% of the patients with MCI and CRF developed dementia, while 40% maintained the same cognitive conditions at the end of the study. Only 32% of the subjects without cardiovascular comorbidities developed dementia. The results of the study suggest that CRF play a key role in cognitive decline of patients with MCI. Patients with MCI and CRF showed not only worse cognitive performances, but also behavioral disorders, depression and functional disability. Patients with CRF had higher conversion rate to AD than the other group, with a mean disease-free period 3 months shorter than the control group.

Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.

A possible role of atrial fibrillation as a risk factor for dementia.

Atrial fibrillation (AF), which is a very common disease among the elderly, is already well known as a risk factor for arterial thromboembolism and stroke. The attention of medical research is now focused on establishing a possible role of AF in the development of cognitive impairment in order to include this arrhythmia among risk factors for dementia. The aim of this work was to investigate the relationship between AF and various types of dementia, such as vascular dementia (VaD), Alzheimer's disease (AD) and mixed dementia (MD). The study consisted of 71 VaD, AD or MD patients, 31 males and 40 females. The sample has been divided in 2 groups according to the sex, and these two groups have been analyzed separately. In females, a statistically significant association was found between mini mental state examination (MMSE) and clinical dementia rating (CDR) scores and AF occurrence (r=-0.32; p<0.05; r=0.33; p<0.05). On the contrary, no significant linear correlation was found between AF and a lower activities if daily living (ADL) and instrumental activities if daily living (IADL) scores. In males, AF/MMSE, AF/CDR, AF/ADL and AF/IADL variables have not been found to be linearly related to each other. Unexpectedly, AF turned to be associated to AD more often than to VAD, becoming a possible risk factor for this neurodegenerative disease. Our results are supported by many studies in literature attributing a basic role of brain hypoperfusion in sporadic AD patho-genesis. More and more scientific data suggest that the already well known risk factors for AD could be considered just the top of an iceberg, providing powerful arguments for impaired cerebral perfusion as the primary trigger in the development of this disease. Moreover, the mildly favorable treatment response in patients with AD to therapy that improves cerebral blood flow is a consistent finding; the same cannot be said of antiamyloid treatments. This opens new possibilities to find an effective way to treat this dramatic pathology.

Mitochondrial DNA sequences in prehistoric human remains from the Alps.

The spread of agriculture that started in the Near East about 10 000 years ago caused a dramatic change in the European archaeological record. It is still unclear if that change was caused mostly by movement of people or by cultural transformations. In particular, there is disagreement on what proportion of the current European gene pool is derived either from the pre-agricultural, paleolithic and mesolithic people, or from neolithic farmers immigrating from the south-east. To begin to characterise the mtDNA gene pool of prehistoric Europe we examined five human remains from the Eastern Italian Alps, dated between 14 000 and 3000 years ago. Three of them yielded sufficient amount of mtDNA for analysis. DNA extracts were prepared in two independent laboratories, and PCR products from the first hypervariable segment of the mtDNA control region were cloned and sequenced. Together with the 5200 year old 'ice man', these DNA sequences show that European mtDNA diversity was already high at the beginning of the neolithic period. All the neolithic sequences have been observed in contemporary Europeans, suggesting genealogical continuity between the neolithic and present-day European mtDNA gene pool. The mtDNA sequence from a 14 000 year-old specimen was not observed in any contemporary Europeans, raising the possibility of a lack of continuity between the mesolithic and present-day European gene pools.

Carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection: the potential is still growing.

This article reviews recent advances of carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Starting from the paper of Dennis C. Johnson [1] in which the great analytical promise of such a technique was anticipated, a multitude of exciting new research possibilities have recently emerged. The great attractiveness of high-performance anion-exchange chromatography is largely due to its compatibility with such a sensitive, selective and reliable detection method as pulsed amperometry. This very good match between liquid chromatography and electrochemical detection has allowed the determination of carbohydrates in a variety of complex matrices, for instance, foods, beverages, diary and biotechnological products, vegetal tissues, and also in the area of clinical diagnostics. For this reason, the introduction of HPAEC-PAD into regulated methods is becoming increasingly accepted. A comprehensive collection of applications to carbohydrates and samples of interest is given, with special focus on the separation of closely related sugar compounds using dilute alkaline eluents. Advances in pulsed potential waveforms are also discussed, and a comparison with other liquid chromatographic methods addressed. 2-keto-3-deoxy-D-glycero-D-galactonononic acid; KDO, 2-keto-3-deoxyoctulosonic acid; FOS, fructooligosaccharides; GF5, GF6, and GF7, oligofructans: Hib, Haemophilus influenzae type b; FAB, fast atom bombardment; ESI, electrospray ionization; MALDI-TOF, matrix assisted laser desorption ionization-time of flight.

Antiandrogens: a summary review of pharmacodynamic properties and tolerability in prostate cancer therapy.

This article provides a summary of the pharmacodynamic properties of major antiandrogens as well as an extensive review of their tolerability. Presently there are two classes of androgen receptor antagonists: the so-called pure, non-steroidal antiandrogens which include flutamide, nilutamide and the more recent bicalutamide and the steroidal antiandrogens cyproterone acetate, megestrol acetate and WIN 49596. Although non steroidal and steroidal compounds have been found to be equally effective in the treatment of prostate cancer presently no studies comparing the use of steroidal or non steroidal antiandrogens with chemical or surgical castration have evaluated quality of life per se. The only advantage of cyproterone acetate on pure antiandrogens seems to be the low incidence of hot flushes; a commonly reported adverse effect of androgen ablative therapy. However, hepatotoxicity associated with long term daily doses of 300 mg daily and the unacceptably high incidence of cardiovascular side effects (10%) should restrict its use to patients who are intolerant of pure antiandrogen compound. In contrast to steroidal compound nonsteroidal compounds let sexual potency to be retained, which is an important consideration with respect to the quality of life of some patients and, at present, the main indication for monotherapy with the pure antiandrogens. As regard as pure antiandrogens clinically important adverse events including gastrointestinal events, particularly diarrhea and occasional disturbances of liver function related to flutamide treatment and antabuse effect, problems with light-dark adaptation and rare interstitial pneumonitis related to nilutamide indicates the bicalutamide, due to its better tolerability profile, together with its once-daily oral administration regimen, could be considered the antiandrogen of first choice in the treatment of prostatic cancer.

Electrooxidation of thiocyanate on the copper-modified gold electrode and its amperometric determination by ion chromatography.

Cyclic voltammetry was used to investigate the electrochemical behavior of an Au/Cu electrode towards the electrooxidation of thiocyanate ion in alkaline medium. The effects of pH, copper loading, scan rate and applied potential on the electrocatalytic oxidation of thiocyanate have been investigated. Flow injection experiments and ion-chromatography (IC) were performed to characterise the electrode as an amperometric sensor for the thiocyanate determination. The effects of carbonate concentration and common interferents on the retention time were also estimated. The electrode stability, precision, limit of detection and linear range were evaluated at a constant applied potential of 0.7 V vs. Ag/AgCl. Calibration plots, obtained in IC, were linear from 1.0 to 195 microM (correlation coefficient of 0.9984). The detection limit (LOD) was 0.5 microM (29 ppb) in a 50 microlitres injection. An example of analytical application, which includes the IC separation and detection of thiocyanate ion present in human urine, is given.

Electrosynthesized non-conducting polymers as permselective membranes in amperometric enzyme electrodes: a glucose biosensor based on a co-crosslinked glucose oxidase/overoxidized polypyrrole bilayer.

A glucose amperometric biosensor based on glucose oxidase immobilized on an overoxidized polypyrrole (PPyox) platinum modified electrode, by glutaraldehyde co-crosslinking with bovine serum albumine, is described. The advantages of covalent immobilization techniques (e.g. high loading and long-term stability of the enzyme) are coupled with the excellent interferent rejection of electrosynthesized non-conducting polymers. The sensor showed an apparent Michaelis-Menten constant of 16 +/- 0.8 mM, a maximum current density of 490 microA/cm2 and a shelf lifetime of at least 3 months. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. Flow-injection response was linear up to 20 mM glucose with typical sensitivity of 84.0 +/- 1.5 nA/mM. The sensor was tested for glucose determination of untreated serum samples from both normal and diabetic subjects; results of amperometric assay compared well with those obtained by a standard enzymatic-colorimetric method.

An Enzyme Switch Employing Direct Electrochemical Communication between Horseradish Peroxidase and a Poly(aniline) Film.

An enzyme switch, or microelectrochemical enzyme transistor, responsive to hydrogen peroxide was made by connecting two carbon band electrodes (∼10 µm wide, 4.5 mm long separated by a 20-µm gap) with an anodically grown film of poly(aniline). Horseradish peroxidase (EC 1.11.1.7) was either adsorbed onto the poly(aniline) film or immobilized in an insulating poly(1,2-diaminobenzene) polymer grown electrochemically on top of the poly(aniline) film to complete the device. In the completed device, the conductivity of the poly(aniline) film changes from conducting (between - 0.05 and + 0.3 V vs SCE at pH 5) to insulating (>+0.3 V vs SCE at pH 5) on addition of hydrogen peroxide. The change in conductivity is brought about by oxidation of the poly(aniline) film by direct electrochemical communication between the enzyme and the conducting polymer. This was confirmed by measuring the potential of the poly(aniline) film during switching of the conductivity in the presence of hydrogen peroxide. The devices can be reused by rereducing the poly(aniline) electrochemically to a potential below +0.3 V vs SCE. A blind test showed that the device can be used to determine unknown concentrations of H(2)O(2) in solution and that, when used with hydrogen peroxide concentrations below 0.5 mmol dm(-)(3), the same device maybe reused several times. The possible development of devices of this type for use in applications requiring the measurement of low levels of hydrogen peroxide or horseradish peroxidase is discussed.