Quantitative determination by screening ELISA and HPLC-MS/MS of microcystins LR, LY, LA, YR, RR, LF, LW, and nodularin in the water of Occhito lake and crops.

The occurrence of harmful cyanobacterial blooms in surface waters is often accompanied by the production of a variety of cyanotoxins, and these toxins are designed to target in humans specific organs on which they act. When introduced into the soil ecosystem by spray irrigation of crops, they may affect the same molecular pathways in plants having identical or similar target organs, tissues, cells, or biomolecules. There are also several indications that terrestrial plants, including crops, can bioaccumulate cyanotoxins and present, therefore, potential health hazards for humans. During this project, for monitoring purposes, water samples were collected from lake Occhito, in which there was an algal bloom (Planktothrix rubescens) in 2009, and from three tanks which acted as hydraulic junctions. In addition, crop samples irrigated with water from the three tanks mentioned above were also picked. Finally, the characterization of principal cyanobacteria was performed, to determine the presence of cyanotoxins such as microcystins and validate a method of screening ELISA for the determination of microcystins in vegetable samples and a confirmatory method by HPLC-ESI-MS/MS. Graphical abstract Occhito lake (left), microcystin LR (center), Tomato field in Foggia (right); figures below: ELISA (left), HPLC-MS/MS (right).

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone.

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.

Cost assessment of headache in childhood and adolescence: preliminary data.

Headache, and in particular migraine, is a common disturbance in childhood and adolescence. The disabling nature of headache, evident in the adult, together with its effects on family life and reduction in performance of scholastic activity, make it a disease with an elevated social economic impact. We present preliminary results of a prospective study conducted over 6 months on a population of headache sufferers in childhood and adolescence who referred to our Juvenile Neuropsychiatry Centre of the Hospital of Perugia. Our objective was to quantify the direct and indirect costs associated with juvenile headache.

Molecular and experimental virulence of Listeria monocytogenes strains isolated from cases with invasive listeriosis and febrile gastroenteritis.

We analyzed 27 Listeria monocytogenes strains of serotypes 1/2b and 4b, from invasive and gastroenteric listeriosis, for molecular and experimental virulence. Molecular virulence was tested by PCR for the presence of 8 major virulence-associated genes and genetic polymorphisms through restriction enzyme analysis; genomic DNA typing using pulsed-field gel electrophoresis was also performed. Experimental virulence was evaluated through intra-peritoneal and intra-gastric mouse virulence assays. Our results showed no significant differences in the virulence-related molecular properties of the strains analyzed. All strains were equally pathogenic following intra-peritoneal inoculation of mice. In mice inoculated intra-gastric with 4 representative strains of the 2 types of listeriosis, there were no significant differences in the bacterial count when comparing invasive and gastroenteric strains, suggesting that the strains were comparable in terms of mean oral infectivity.

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains.

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.

Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells.

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.