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Canine Heartworm Disease, caused by Dirofilaria immitis, primarily affects canids and felids. The earliest studies on cryopreservation were carried out at -70°C, achieving acceptable survival rates, however microfilariae (mf) showed alterations both in morphology and motility. Thereafter, liquid nitrogen was used representing an excellent tool for long-term preservation, albeit it is expensive and requires trained personnel. Accordingly, the aim of this study was to develop a protocol for cryopreservation of D. immitis mf at -80°C feasible to laboratories with limited specialized equipment. The cryoprotectant medium was composed by 5 % dimethyl sulfoxide, 20 % of newborn calf serum and 75 % of saline solution. At Study Day (SD) 0 whole blood from a D. immitis naturally infected dog was diluted with the medium at a ratio of 1:1 and stored at -80°C using a freezing container (Nalgene® Mr. Frosty® Cryo 1°C). On the SD1 and then once a month, one cryovial was thawed and examined for survival, motility, length and morphology of mf. On SD 1, the mf showed a survival rate of 99 %. By SD 120 the survival rate gradually decreased (up to 63 %) and a shift in motility patterns between the "medium" and "slow" classes, was observed. On SD 150, the survival rate exceeded 50 % and mf did not exhibit detectable morphological alterations; however, a reduction in length was observed. This study marks the first protocol where the -80°C freezer has been employed for cryopreservation of D. immitis, integrating the application of cryoprotectants and novel techniques for gradual temperature transition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: To achieve success in endodontic treatment, it is essential to properly perform the steps of shaping, cleansing and obturation. Determining the working length of the canal is, therefore, a process that must be precise and accurate. Electronic apex locators are a useful tool for the clinician to best perform this step of endodontic treatment. Materials and Methods: The purpose of the following umbrella review is to evaluate, through data in the literature, the degree of accuracy of apex locators. Results: Seven systematic reviews were included in the following umbrella review. Five compare the accuracy of apex locators versus radiographic techniques, two compare different types of electronic apex locators, and two analyze the determination of working length in primary teeth. Conclusions: From the results obtained from the following umbrella review, albeit at low levels of evidence, the methods for determining working length using electronic apex locators and other methods, particularly using radiographic evaluation, are equally valid.

Dirofilaria repens (Spirurida: Onchocercidae) is the causative agent of canine subcutaneous dirofilariosis. Adults of D. repens localize in the subcutaneous tissues and fasciae and most of the infected animals are asymptomatic though dermatological disorders have been described sporadically. In this study, the gonads of 100 male dogs were inspected for D. repens presence from December 2020 to January 2022. All examined dogs were older than 48 months and underwent castration for several reasons. The testes and the superficial layers were examined with the aid of stereomicroscope; in addition, the blood of all included dogs was collected and analysed by Knott's test to assess the presence of microfilariae. Six out of 100 (6 %, CI95%, 2.2-12.6 %) examined dogs were positive for D. repens presence, and in all the cases the parasites were found beneath the tunica vaginalis layers. Microfilariae were found in 15 blood samples including 5 out of those 6 testes positive dogs thus resulting in a final D. repens prevalence of 16 % (CI95%: 9.4-24.7 %). No pathological changes and or lesions due to parasite presence were observed in the testes or in their layers. The present study represents the first survey focusing on the detection of D. repens in dog testes on a large sample size. Adult parasites were found in the testes of 6 out of 16 D. repens infected dogs accounting for 37.5 % of cases and suggesting as testicular localization is not uncommon. The absence of neoplastic or inflammatory lesions in D. repens infected testes suggests as these conditions do not act as attractive stimuli for the parasite localization. Finally, considering the zoonotic potential of D. repens and the large infection prevalence here reported, active surveillance in endemic areas is strongly advocated to limit the presence of reservoir and prevent further infections to animals and humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: A Special Health Care Need (SHCN) is characterized by any type of physical, mental, sensorial, cognitive, emotional, or developmental condition that requires medical treatment, specialized services, or healthcare interventions. These conditions can negatively impact oral health as SHCN children can hardly cooperate or communicate and experience higher levels of dental fear/anxiety, which interfere with regular appointments. The present narrative review aims to analyze the use of audiovisual (AV) tools in dental setting for the management of SHCN children during dental treatment and to evaluate their effectiveness in anxiety/behavior control from the child, dentist, and care-giver perspectives. This analysis leads to the proposal of a new multi-session model for the behavioral management of SHCN pediatric subjects., Methods: An electronic search on the MEDLINE/Pubmed, Scopus, and Web of Science databases was carried out and through this analysis, a new model was proposed, the "UNISA-Virtual Stepwise Distraction model", a multi-session workflow combining traditional behavior management and the progressive introduction of AV media to familiarize the SHCN child with dental setting and manage behavior., Results: AV tools helped in most cases to manage SHCN behavior and decreased stress in both the dentist and child during dental treatments. Care-givers also welcomed AV distractors, reporting positive feedback in using them during future treatments., Conclusions: The present narrative review found increasing evidence of the use of AV media for SHCN pediatric subjects as distraction tools during dental treatment. In the majority of the studies, AV tools proved to be effective for the management of anxiety, dental fear, and behavior in dental setting.

Reconfiguration of chemical sensors, intended as the capacity of the sensor to adapt to novel operational scenarios, e.g., new target analytes, is potentially game changing and would enable rapid and cost-effective reaction to dynamic changes occurring at healthcare, environmental, and industrial levels. Yet, it is still a challenge, and rare examples of sensor reconfiguration have been reported to date. Here, we report on a reconfigurable label-free optical sensor leveraging the versatile immobilization of metal ions through a chelating agent on a nanostructured porous silica (PSiO 2 ) optical transducer for the detection of different biomolecules. First, we show the reversible grafting of different metal ions on the PSiO 2 surface, namely, Ni 2+ , Cu 2+ , Zn 2+ , and Fe 3+ , which can mediate the interaction with different biomolecules and be switched under mild conditions. Then, we demonstrate reconfiguration of the sensor at two levels: 1) switching of the metal ions on the PSiO 2 surface from Cu 2+ to Zn 2+ and testing the ability of Cu 2+ -functionalized and Zn 2+ -reconfigured devices for the sensing of the dipeptide carnosine (CAR), leveraging the well-known chelating ability of CAR toward divalent metal ions; and 2) reconfiguration of the Cu 2+ -functionalized PSiO 2 sensor for a different target analyte, namely, the nucleotide adenosine triphosphate (ATP), switching Cu 2+ with Fe 3+ ions to exploit the interaction with ATP through phosphate groups. The Cu 2+ -functionalized and Zn 2+ -reconfigured sensors show effective sensing performance in CAR detection, also evaluated in tissue samples from murine brain, and so does the Fe 3+ -reconfigured sensor toward ATP, thus demonstrating effective reconfiguration of the sensor with the proposed surface chemistry.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Psoriasis, a systemic autoimmune disorder primarily affecting the skin, manifests through erythematous plaques and scales, impacting approximately 2-3% of the global population. Chronic periodontitis, a prevalent oral disease characterized by the destruction of tooth-supporting tissues, affects roughly 10-15% of adults worldwide. Emerging evidence suggests a bidirectional relationship between psoriasis and chronic periodontitis, supported by epidemiological studies indicating a higher prevalence of periodontitis among individuals with psoriasis and vice versa. Both conditions are chronic inflammatory diseases marked by dysregulated immune responses and altered cytokine profiles, notably involving proinflammatory cytokines such as TNF-α and IL-17. Clinical studies highlight a reciprocal impact of treating one condition on the other, underscoring the necessity of interdisciplinary collaboration between dermatologists and periodontists in managing patients with both conditions. This narrative review provides a comprehensive overview of the relationship between psoriasis and chronic periodontitis, examining epidemiological associations, shared inflammatory pathways, genetic insights, microbial dysbiosis, environmental factors, and clinical implications. The review emphasizes the importance of integrated care approaches and the potential for targeted therapeutic interventions to improve both psoriatic and periodontal patient outcomes, advocating for further research into the molecular and cellular mechanisms underpinning the comorbidity of these diseases.

Background and Objectives: The present systematic review aims to assess the success rate of the pulp regeneration treatment, according to the American Association of Endodontists (AAE) criteria, using different bioactive materials in permanent teeth of pediatric subjects (6-17 years of age)., Materials and Methods: The study protocol was registered on PROSPERO and adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement. The question formulation was accomplished using the PICO model, and an electronic search was carried out on Scopus, MEDLINE/PubMed, Web of Science, and Cochrane databases till April 1, 2023. A total of 30 studies were established to fulfill the inclusion criteria of this systematic review., Results: A total of 273 teeth have been treated with pulp regeneration treatment. By comparing different biomaterials and the success criteria defined by the AAE, the material associated with a higher success rate was found to be the white mineral trioxide aggregate. However, the overall success rate of pulp regeneration treatment was reported for 248 out of 273 teeth (91.20%)., Conclusions: Data obtained support the potential that regenerative endodontics aids in continuing root development in permanent immature teeth. Further studies are needed for a more extensive evaluation of the use of different biomaterials and the success rate in regenerative endodontics., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Conservative Dentistry and Endodontics.)

The saddled seabream, Oblada melanura (L.), is a common seawater species present in the Mediterranean. Between July and August 2023, during diving activities along the Sicilian coast of Italy, we recorded with a digital camera several specimens of O. melanura showing an abnormal pattern of swimming in the water column. The unusual swimming behaviour was characterized by fast, uncoordinated directional changes and isolation from the remaining fishes in the shoal. Four dead fish were found and collected for necropsy and parasitological evaluation/examination. Upon gross examination, all fish showed an evident coelomic distension; the coelomic cavity of two fish was filled with nematodes that dislocated the coelomic organs, and the other two had degraded nematodes and a conspicuous quantity of fluid in the coelomic cavity. All collected parasites were identified as Philometra obladae (Nematoda: Philometridae) according to morphological criteria. Here, we describe the unusual swimming behaviour of O. melanura naturally infected with Ph. obladae and the results of the examination of dead fish infected with this parasite. Future studies are needed to better evaluate and describe the dynamics and the epidemiology of Ph. obladae infection in wild O. melanura ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Twaite shad ( Alosa fallax ) includes two subspecies, the anadromous Alosa fallax nilotica , and the landlocked species Alosa fallax lacustris , representing the only Clupeidae inhabiting Lake Garda. Study of the parasitic fauna of this species in this area is still limited. For this reason, the aim of the present study was to evaluate the presence of tapeworms from A. fallax lacustris sampled in Lake Garda. Sixty-six A. fallax lacustris specimens were collected at the Milan fish market (Lombardy, Italy); specifically, an evaluation of the gastrointestinal contents was carried out for the presence of helminths. All parasites found were fixed in 70% ethanol and transferred to the laboratories of the University of Messina for morphological and molecular analysis. Parts of the parasites were stained with Semichon's carmine red technique. Molecular analysis was carried out using LSU rRNA and ITS2 region genes. Eight specimens out of sixty-six (12.1%) were positive for adult cestodes within the pyloric caeca. Morphological and molecular analysis could to identify the parasites found to be Proteocephalus longicollis . This parasite species is not considered a zoonotic agent, representing a low risk of parasitic fish-borne zoonosis for consumers of this appreciated fish from Lake Garda.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

Bluntnose sixgill shark, Hexanchus griseus (Bonnaterre, 1788), is a little-known elasmobranch in the Mediterranean Sea. Given the lack of information about H. griseus , the aim of our study was to describe the helminth fauna of this species. In March 2023, one H. griseus juvenile female specimen was found off the coast of Messina (Italy) and referred by the Italian Coast Guard to our laboratory for necropsy and parasitological evaluation. After necropsy, the specimen's gills, stomach and spiral valve were investigated for parasite presence. All collected parasites were stored in 70% ethanol for routine parasitological analysis. No lesions due to parasites were found in the gills or skin. Three species of helminths were found in one studied female specimen of Hexanchus griseus , namely, two cestode species ( Phyllobothrium sinuosiceps and Nybelinia sp., larvae) and one trematode ( Otodistomum veliporum) . Among them, five Trypanorhyncha plerocercoid larvae were found attached to the stomach mucosa, and six adult cestodes and one digenean trematode were collected from the spiral valve. No other parasite taxa were found in the celomic organs. This study reports new information regarding the parasitic fauna of H. griseus from the central Mediterranean Sea.

In recent decades, the number of autochthonous cases and foci of Dirofilaria immitis in dogs from southern regions has increased considerably, suggesting that the distribution of the species is not limited to northern Italian regions. This epidemiological picture emerges from case reports or studies in specific locations where outbreaks of heartworm disease have occasionally been reported together with the presence of mosquito vectors. To obtain a more comprehensive picture of the current distribution of D . immitis in southern Italy, a multicenter cross-sectional survey of canine filariasis was conducted. Owned and sheltered dogs ( n  = 1,987) were included in the survey regardless their breed, attitude and/or sex. All included dogs were older than 1 year and had no history of chemoprophylactic treatment against filarioses. A blood sample was collected from enrolled dogs and screened by modified Knott's test and, when positive, tested using D . immitis specific ELISA rapid test (SNAP 4DX, IDEXX). The overall microfilaremia prevalence was 17% ( n  = 338) being single-species infection (92.6%) more common that mixed (7.4%). Remarkably, D . immitis was the most frequent species detected with an overall prevalence of 11.4% ( n  = 227), followed by Dirofilaria repens ( n  = 74; 3.7%), and Acanthocheilonema reconditum ( n  = 12; 0.6%). Sheltered dogs were significantly more infected by D . immitis , as well as mongrel dogs and animals housed in rural areas. Data here reported indicate that D . immitis is largely present in southern Italy, raising awareness about the necessity of proper screening and chemoprophylactic treatments in exposed animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Napoli, De Benedetto, Ciuca, Bosco, Lia, Veneziano, Bezerra Santos, Otranto, Rinaldi and Brianti.)

Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host-parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus ( n = 99), Chelon auratus ( n = 37), and Oedalechilus labeo ( n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at -80 °C for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites ( Neoechinorhynchus agilis ) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes ( C. labrosus , 49.5 %; C. auratus , 27%, and O. labeo , 50%), molecularly identified as Haploporus benedeni . This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon.

This study represents the first report of Hysterothylacium incurvum within swordfish ( Xiphias gladius ) heart chambers. Swordfish is a large pelagic teleost, considered one of the most appreciated fish worldwide. Among swordfish parasites, Anisakis sp. and Hysterothylacium sp. have been used to evaluate biological and ecological aspects of this teleost. Between 2021 and 2022, 364 X. gladius hearts, caught from the Atlantic Ocean (FAO 27.IXa and FAO 34 areas), were collected at the Milan fish market (Lombardy, Italy). Three specimens from FAO 27.IXa was positive for seven adult nematodes ( p = 1.55%) within the heart chambers. Of these, three specimens were found within the bulbus arteriosus and 4 in the ventricle. All parasites were stored in 70% ethanol and processed for parasitological and molecular analysis using Cox2 , ITS regions/ITS-I-5.8S-ITS-II, and rrnS genes. The analysis allowed us to identify the retrieved parasite as H. incurvum . According to our evaluation, the final localization is due to the movement of L3 larvae from the coelomic cavity to the bloodstream, with consequent development to the adult stage within the heart. Finally, the parasite localization, considered non-marketable fish parts, does not pose a significant risk to consumers, also considering the low zoonotic potential of H. incurvum .

The current study provides a morphological and molecular characterization of a new species of Didymodiclinus (Trematoda: Didymozoidae) infecting the dusky grouper, Epinephelus marginatus (Teleostei: Serranidae) from the Mediterranean Sea. A total of 279 dusky grouper specimens were examined for didymozoid gill parasites from the Mediterranean Sea between 1998 and 2020. New species differs from the most similar congeneric species by the rudiments of female reproductive organs in functional male specimens, and the seminal receptacle, Mehlis gland and accessory gland cells in functional female specimens, not observed in Didymodiclinus branchialis (Yamaguti, 1970), Didymodiclinus epinepheli (Abdul-Salam, Sreelatha and Farah, 1990) and Didymodiclinus pacificus (Yamaguti, 1938), respectively. These species are also characterized by their different hosts and location within the host tissues, being from other geographical localities. Moreover, this is the first species reported in E. marginatus from the central and western Mediterranean Sea. Genetic analyses were performed on partial 28S and partial internal transcribed spacer-2 ribosomal RNA regions and the mitochondrial cytochrome oxidase 1 (cox1) gene by polymerase chain reaction. Comparison of genetic sequences of Didymodiclinus marginati n. sp. with the available deposited sequences of 28S revealed that the new isolates cluster with several unidentified didymozoids and groups as a sister clade of the Nematobothrinae subfamily. Moreover, 28S and cox1 phylogenetic trees evidenced that Didymodiclinae is well separated from Didymozoinae and other gonochoric didymozoids. Following both morphological and genetic results, a key of identification for the genus Didymodiclinus is proposed.

The response to allopurinol treatment and survival time of a case of feline leishmaniosis in a FIV co-infected cat is herein reported. In May 2019, a 13-year-old neutered European Shorthair male was referred due to weight loss and exfoliative dermatitis. Lymphadenomegaly and splenomegaly were detected upon clinical inspection, while the presence of several Leishmania infantum amastigotes were detected on splenic and lymphonodal fine needle aspiration (FNA). Allopurinol (10 mg/kg PO q 12 h) was administered. After two months, the cat's clinical symptoms disappeared, and the owners decided to interrupt the therapy. In February, two reddish nodular fleshy neoformations appeared in both eyes, and amastigotes of Leishmania were detected by cytology on conjunctival swabs. Allopurinol treatment was re-started at the same rate; the ocular lesions regressed in two weeks, and the owner again decided to interrupt the therapy. In July, the patient had a new relapse, but the owner, tired of continuous relapses, refused further treatment of the disease. The cat's health condition continued to worsen: in October 2021, the ocular lesions appeared again, and in November the patient died. This case underlines the effectiveness of allopurinol and highlights how interruption of treatment frequently leads to relapse, impairing the animal's health condition and prognosis.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioids of zoonotic concern, which primarily cause canine heartworm disease (HWD) and subcutaneous dirofilariosis, respectively. The geographical distribution of these nematodes is constantly changing, due to many factors such as the colonization by new invasive mosquito species, the increased movement of animals and the absence of chemoprophylactic strategies in non-endemic regions. Although HWD has always been considered endemic in northern Italy, an increase in the prevalence of this disease has been recorded in the last decades in central and southern regions. We describe the southernmost hyperendemic European focus of heartworm disease in the Pelagie archipelagos. From June to November 2020, 157 dogs and 46 cats were sampled in Linosa and Lampedusa islands for the detection of Dirofilaria immitis and Dirofilaria repens by modified Knott's, rapid antigen and molecular tests. Mosquitoes were collected using CDC light and BG sentinel-2 traps and aspirators, and tested for host blood meal and Dirofilaria spp. DNA. Out of 56 dogs in Linosa and 101 in Lampedusa, 33 (i.e. 58.9%) and 8 (i.e. 7.9%) were positive to D. immitis and D. repens, respectively. Three cats scored positive to D. immitis (i.e. 17.6%) in Linosa. Six mosquito species were identified, and the abdomen of a non-engorged Aedes albopictus was positive for D. immitis and human DNA. The results suggest that D. immitis infection could spread to new previously non-endemic territories in southern Europe, representing a real threat to animal and human health., (© 2021 Wiley-VCH GmbH.)

The aim of the present study was to describe gastric granuloma caused by Anisakis pegreffii in Sphyraena viridensis caught in the central Mediterranean Sea. Sixty-eight S. viridensis specimens were collected from different fish markets on the east coast of Sicily. Coelomic organs were observed both macroscopically and with the aid of stereomicroscope. Parasite specimens and lesioned tissues were collected for identification, histological and molecular analyses. Twelve specimens ( p = 17.6%) were positive for the presence of nematode larvae, morphologically identified as larvae of Anisakis sp., with values of mean abundance and mean intensity of 0.9 and 4.8, respectively. One large female specimen showed massive parasite infection associated with nodular lesions of the gastric wall. By histology, several nematode larvae encysted through the gastric wall were found. The parasite bodies were surrounded by a granulomatous reaction made up of macrophages, epithelioid cells, some lymphocytes and an external connective sheet. Molecular analysis of 18S rRNA and cox2 genes from Anisakis sp. collected larvae, identified them as A. pegreffii . The lesions here described, though macroscopically superimposable on human eosinophilic granuloma, microscopically showed significant differences in the inflammatory cells involved and in the type of immune reaction.

Bovine besnoitiosis is a debilitating infectious disease caused by Besnoitia besnoiti (Apicomplexa; Sarcocystidae). The disease is mainly characterized by cutaneous and systemic signs, infertility in bulls, and abortion in cows. The current study describes an autochthonous outbreak of bovine besnoitiosis in Sicily, Southern Italy, being the first report of B. besnoiti infection in the island so far. In a cattle farm located in Syracuse province, a 4-year-old Belgian blue bull born in Sicily displayed typical clinical signs of the sub-acute/chronic disease phase with thickening of the skin of the scrotum and testicular degeneration. Histological examination of scrotal biopsies revealed the presence of several tissue cysts of B. besnoiti. The serological analysis of the herd using a commercial ELISA revealed a high seroprevalence (45 out of 55; 82%) of antibodies against B. besnoiti. Few seropositive animals (5 out of 45; 11%) showed clinical signs, cysts in vestibulum vaginae (1 out of 31; 3.2%), and testicular degeneration (4 out of 14; 28.6%) assessed by ultrasonographic investigations. The paucity of clinical signs associated with the high seroprevalence in the farm led to hypothesize that bovine besnoitiosis is endemic in the area though further studies are needed. Local practitioners should be more aware of the disease to facilitate the early detection of cases, prevent the spread of infection, and avoid economic losses and animal health problem., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study aimed to investigate parasite fauna of E. marginatus from the central Mediterranean Sea between Messina and Syracuse. In the present survey; parasite fauna of dusky grouper was investigated for two main reasons: the economic value of this species and the current lack of studies regarding the capture area. Seventy dusky groupers were caught from May 2018 to February 2020. Forty-seven out of the 70 specimens (67.2%) were infected with one or more parasite species. The most abundant species was Prosorhynchus caudovatus (42.9%), followed by Podocotyle temensis (28.6%), Didymodiclinus sp. (18.6%), Philometra jordanoi (5.7%), Anisakis Type II larvae (5.7%). Higher prevalence of infection of P. jordanoi and Contracaecum sp. was found in warm months (March to September), while P. caudovatus and P. temensis were mostly found during cold months. Weight and total length of E. marginatus were positively correlated with the parasitic load of P. jordanoi and Didymodiclinus sp. The different prevalence of parasite infection found between warm and cold months is probably related to the diet of the dusky grouper; which is characterized by mollusks that are intermediate hosts for parasite species found. None of the parasites found in the present survey is responsible for zoonosis.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

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.

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.

Herein we present a straightforward approach for the use of polydopamine (PDA) in adsorption of heavy metals from aqueous solutions. This is achieved by fabricating a healthy and environmentally friendly polydimethylsiloxane (PDMS) foam with a mussel inspired PDA layer deposited on the surface. Critical adsorption parameters (pH, temperature and PDA thickness) are optimized by the application of experimental design methodology. Adsorption kinetics and isotherms are studied in detail evidencing a good fitting with Langmuir isotherm and pseudo-second-order kinetics thus suggesting the occurrence of a chemical sorption process with monolayer nature between metals and PDMS/PDA foam. Intraparticle diffusion model evidences good accessibility and high affinity of binding sites on PDA surface. Once adsorbed, metals are reduced to a lower toxic form and can be then removed by a mild acidic treatment thus being easily collected and stocked., (Copyright © 2019 Elsevier Inc. All rights reserved.)

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM 197 , a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of 1 H- 1 H and 1 H- 13 C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this work, we explore the effects of O-acetylation on the physical and immunological characteristics of the WHO International Standards of Vi polysaccharide (Vi) from both Citrobacter freundii and Salmonella enterica serovar Typhi. We find that, although structurally identical according to NMR, the two Vi standards have differences with respect to susceptibility to de-O-acetylation and viscosity in water. Vi standards from both species have equivalent mass and O-acetylation-dependent binding to a mouse monoclonal antibody and to anti-Vi polyclonal antisera, including the WHO International Standard for human anti-typhoid capsular Vi PS IgG. This study also confirms that human anti-Vi sera binds to completely de-O-acetylated Vi. Molecular dynamics simulations provide conformational rationales for the known effect of de-O-acetylation both on the viscosity and antigenicity of the Vi, demonstrating that de-O-acetylation has a very marked effect on the conformation and dynamic behavior of the Vi, changing the capsular polysaccharide from a rigid helix into a more flexible coil, as well as enhancing the strong interaction of the polysaccharide with sodium ions. Partial de-O-acetylation of Vi revealed hidden epitopes that were recognized by human and sheep anti-Vi PS immune sera. These findings have significance for the manufacture and evaluation of Vi vaccines., (Copyright © 2019. Published by Elsevier Ltd.)

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage. We developed a conjugation chemistry for linking O-antigen to CRM 197 carrier protein, through sequential insertion of adipic acid dihydrazide (ADH) and adipic acid bis( N-hydroxysuccinimide) ester (SIDEA) as linkers, without impacting O-antigen chain epitopes. Here the resulting sugar-protein connectivity has been investigated in detail. The core portion of the lipopolysaccharide was used as a model molecule to prepare CRM 197 conjugates, making structural investigations easier. The first step of reductive amination with ADH involves the terminal 3-deoxy-d- manno-oct-2-ulosonic acid (KDO) residue of the core region. The second reaction step resulted not to be selective, as SIDEA reacted with both ADH and pyrophosphorylethanolamine (PPEtN) of the core region, independently from the pH at which the reaction was performed. Peptide mapping analysis of the deglycosylated core-CRM 197 conjugates confirmed that lysine residues of CRM 197 were linked to SIDEA not only through KDO-ADH but also through PPEtN. This analysis also confirmed that the conjugation chemistry is random on the protein, involving a large number of lysine residues, particularly the surface exposed ones. The method for core-CRM 197 characterization was successfully extended to O-antigen-CRM 197 conjugate, confirming the results obtained with the core. This study not only allowed full characterization of OAg-CRM 197 conjugates, but can be applied to optimize synthesis and characterization of other OAg-based glycoconjugate vaccines. Analytical methods to investigate saccharide-protein connectivity are also of fundamental importance to study the relationship between glycoconjugate structure and immune response induced.

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.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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 PM 10 . 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 PM 2.5 and PM 10 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 PM 2.5 and 12% of PM 2.5-10 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: NaNO 3 , correlated with chloride depletion and aging of sea-spray, mainly present in PM 2.5-10 ; NH 4 NO 3 more abundant in PM 2.5 . 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the last years, outer membrane vesicles have attracted a lot of attention for the development of vaccines against bacterial pathogens. Extracellular vesicles can be obtained in high yields by genetic mutations, resulting in generalized modules for membrane antigens (GMMA). Methods to check the quality, consistency of production, and stability of GMMA vaccines are of fundamental importance. In this context, analytical methods for size distribution determination and verifying the integrity and possible aggregation of GMMA particles are strongly needed. Herein, GMMA particle size distribution has been evaluated by means of three different techniques. Dynamic light scattering (DLS), multiangle light scattering (MALS) coupled with high-performance liquid chromatography-size exclusion chromatography (SEC), and nanoparticle tracking analysis (NTA) have been compared to characterize GMMA from different mutants of Salmonella typhimurium and Salmonella enteritidis strains. We found that the presence of O-antigen chains on GMMA determined higher Z -average diameters by DLS compared to size estimation by MALS and that the hydrodynamic diameter increased with the number of O-antigen chains per GMMA particle. In the case of SEC-MALS, the size of the whole population better reflects the size of the most abundant particles, whereas DLS diameter is more influenced by the presence of larger particles in the sample. SEC-MALS and NTA are preferable to DLS for the analysis of bimodal samples, as they better distinguish populations of different size. MALS coupled to a size exclusion chromatography module also allows checking the purity of GMMA preparations, allowing determination of generally occurring contaminants such as soluble proteins and DNA. NTA permits real-time visualization with simultaneous tracking and counting of individual particles, but it is deeply dependent on the choice of data analysis parameters. All of the three techniques have provided complementary information leading to a more complete characterization of GMMA particles., Competing Interests: The authors declare the following competing financial interest(s): Francesca Micoli and Carlo Giannelli are employees of GSK Vaccines Institute for Global Health (GVGH), part of the GSK group of companies. Gianluigi De Benedetto participated in a postgraduate studentship program at GSK Vaccines.

Based on the motivational sequence described in Self-Determination Theory, this study explored the relationship between relatedness, motivation, adaptation and leisure in nursing homes. We formulated the hypothesis that the variables of the study would be found in an integrative mediational sequence: Participation in leisure activities→Relatedness→Self-determined motivation→Adaptation to nursing homes. Participants (N=112, mean age=84.17) were invited to complete questionnaires assessing these variables. Results of the path analysis found an unsatisfactory fit for this model but revealed another model (Model 2) with a good fit index: Relatedness→Participation in leisure activities→Self-determined motivation→Adaptation to nursing homes→Relatedness. Model 2 fitted better than model 1: the Chi-square values were not significant, Chi 2 (df=2)=5.1, p=0.078 and other indices were satisfactory (CFI=0.930, RMSEA=0.049 and NFI=0.918). These results suggest that feeling connected and secure in the relationships with others, and integrated as an individual to the group contribute to enhance leisure practice, self-determined motivation, and finally adaptation to life environment. Consequently, the relatedness promotes leisure activities practice which represents a central adaptive behavior in nursing homes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2016. Published by Elsevier Ltd.)

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Klebsiella pneumoniae are Gram negative opportunistic pathogens producing capsular (K) polysaccharides. Seventy-seven different K antigens have been described and they are the basis for K serotyping. Capsular polysaccharides are important virulence factors and have a relevant role for the structure of biofilm communities. Nevertheless, little information is available on the polysaccharides produced in biofilm matrices by Klebsiella spp. In the present study, a clinical isolate of Klebsiella pneumoniae was grown both on cellulose membranes deposited on agar plates, where it formed an adherent biofilm, and in liquid medium, where it formed floating biofilms (flocs). Extraction and purification of the polysaccharide fraction showed that only one main carbohydrate polymer was present in both adherent biofilms and flocs. Composition and linkage analysis, Smith degradation followed by ESI-MS, 1D and 2D NMR spectroscopy revealed that the polysaccharide belong to the type K24 and has the following structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Largely used as a natural biological tag in studies of dispersal/connectivity of fish, otolith elemental fingerprinting is usually analyzed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). LA-ICP-MS produces an elemental fingerprint at a discrete time-point in the life of a fish and can generate data on within-otolith variability of that fingerprint. The presence of within-otolith variability has been previously acknowledged but not incorporated into experimental designs on the presumed, but untested, grounds of both its negligibility compared to among-otolith variability and of spatial autocorrelation among multiple ablations within an otolith. Here, using a hierarchical sampling design of spatial variation at multiple scales in otolith chemical fingerprints for two Mediterranean coastal fishes, we explore: 1) whether multiple ablations within an otolith can be used as independent replicates for significance tests among otoliths, and 2) the implications of incorporating within-otolith variability when assessing spatial variability in otolith chemistry at a hierarchy of spatial scales (different fish, from different sites, at different locations on the Apulian Adriatic coast). We find that multiple ablations along the same daily rings do not necessarily exhibit spatial dependency within the otolith and can be used to estimate residual variability in a hierarchical sampling design. Inclusion of within-otolith measurements reveals that individuals at the same site can show significant variability in elemental uptake. Within-otolith variability examined across the spatial hierarchy identifies differences between the two fish species investigated, and this finding leads to discussion of the potential for within-otolith variability to be used as a marker for fish exposure to stressful conditions. We also demonstrate that a 'cost'-optimal allocation of sampling effort should typically include some level of within-otolith replication in the experimental design. Our findings provide novel evidence to aid the design of future sampling programs and improve our general understanding of the mechanisms regulating elemental fingerprints.

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Information about dispersal scales of fish at various life history stages is critical for successful design of networks of marine protected areas, but is lacking for most species and regions. Otolith chemistry provides an opportunity to investigate dispersal patterns at a number of life history stages. Our aim was to assess patterns of larval and post-settlement (i.e. between settlement and recruitment) dispersal at two different spatial scales in a Mediterranean coastal fish (i.e. white sea bream, Diplodus sargus sargus) using otolith chemistry. At a large spatial scale (∼200 km) we investigated natal origin of fish and at a smaller scale (∼30 km) we assessed "site fidelity" (i.e. post-settlement dispersal until recruitment). Larvae dispersed from three spawning areas, and a single spawning area supplied post-settlers (proxy of larval supply) to sites spread from 100 to 200 km of coastline. Post-settlement dispersal occurred within the scale examined of ∼30 km, although about a third of post-settlers were recruits in the same sites where they settled. Connectivity was recorded both from a MPA to unprotected areas and vice versa. The approach adopted in the present study provides some of the first quantitative evidence of dispersal at both larval and post-settlement stages of a key species in Mediterranean rocky reefs. Similar data taken from a number of species are needed to effectively design both single marine protected areas and networks of marine protected areas.

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., (Copyright 2009 Elsevier B.V. All rights reserved.)

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.

Glycoalkaloids are naturally occurring nitrogen-containing compounds present in many species of the family Solanaceae, including cultivated and wild potatoes (Solanum spp.), tomatoes (Lycopersicon spp.), etc. These compounds have pharmacological and toxicological effects on humans due to their significant anticholinesterase activity and disruption of cell membranes. Herein is reported the development of a capillary electrophoresis (CE) method using nonaqueous (NA) separation solutions in combination with ion trap mass spectrometry (MS and MS/MS) detection for the identification and quantification of glycoalkaloids and their relative aglycones. A mixture 90:10 v/v of MeCN-MeOH containing 50 mM ammonium acetate and 1.2 M acetic acid (applied voltage of 25.5 kV) was selected as a good compromise for the separation and detection of these compounds. The electrospray MS measurements were carried out in the positive ionization mode using a coaxial sheath liquid, methanol-water (1:1) with 1% of acetic acid at a flow rate of 2.5 microL/min. Under optimized experimental conditions, the predominant ion was the protonated molecular ion ([M+H](+)) of solanidine (m/z = 398), tomatidine (m/z = 416), chaconine (m/z = 852), solanine (m/z = 868), and tomatine (m/z = 1034). MS/MS experiments were carried out systematically by changing the relative collisional energy and monitoring the intensities of the fragment ions that were not high enough to allow better quantification than with the mother ions. The method was used for analyzing glycoalkaloids in potato extracts.

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.

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.

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.

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.

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.