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L’entomologia forense, come ormai ampiamente noto, viene utilizzata routinariamente nelle indagini forensi, in particolare in quei casi nei quali i classici dati tanatocronologici (ipostasi, rigidità, temperatura corporea, ecc.) non possono essere d’ausilio per l’identificazione dell’epoca della morte. Gli insetti campionati su resti cadaverici, per potere essere utilizzati a fini forensi, necessitano di una precisa identificazione di specie, dato che ciascuna specie si caratterizza per una differente durata del ciclo vitale. Gli insetti che colonizzano il cadavere, soprattutto nelle prime fasi di colonizzazione, hanno una distribuzione abbastanza uniforme in tutto il mondo, tuttavia è possibile a volte rinvenire delle differenze correlate alla fauna locale. In questo lavoro viene presentato un case report in cui dimostriamo l’utile integrazione tra i dati di entomologia forense e quelli di tassonomia molecolare.

In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/ degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.

The present invention relates to the production of recombinant collagenases, and in particular describes a method for the production of recombinant clostridium histolyticum collagenases Col characterized by a yield higher than approximately 140 mg/l of culture of said collagenases in soluble, biologically active form, collagenases produced by this method, compositions comprising these collagenases and the use thereof.

In small intestinal disease, coeliac sprue, proline-rich gluten peptides from wheat, rye and barley are relatively resistant to gastrointestinal digestion, and therefore remain in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. Since most serine endopeptidases are unable to hydrolyse proline residues, proline specific proteases may be therapeutic keys in digestive diseases. Partial hydrolysis reduces the risk of allergenic sensitization while total hydrolysis ensures the elimination of the allergenicity of whey protein (Villad´oniga and others 2007). Kimoto and others (1998) reported that 18-, 31-, 37- and 58-kDa wheat allergens were recognized by the IgE antibodies in the sera of 20%, 60%, 60%, and 57% of allergic patients, respectively. Gliadin has been considered to cause wheat-dependent exercise-induced anaphylaxis. In this work, we used a new mixture of extracted marine proteases to digest gliadin. The enzymes were extracted from epatopancreas of Eriphia verrucosa and Palinurus elephas; the tissues were homogenised and after differential centrifugation the enzymes were used in enzymatic assay. The digestion was performed at low temperature (4°C). After digestions the substrate was analysed in SDS-PAGE and the allergenic fragments result are completely digested by the action of both different marine species enzymes. At present, we are try to identify the marine prolyl endopeptidases and/or prolyl oligopeptidases, families of serine proteases having the ability to hydrolyse the peptide bond on the carboxyl side of a proline residue. These proline specific enzymes are widely distributed in bacteria, fungi, animals and plants.

L'Analisi dei polimorfismi del DNA tra le metodologie di identificazione dei bovini. Analisi dei rapporti di filiazione in un'allevamento italiano. In 22° Convegno Nazionale Genetisti Forensi italiani.

Role of E-selectin in the modulation of metastatic phenotype

ANALISI DI POLIMORFISIMI STR NELLA SICILIA OCCIDENTALE.

UNA NUOVA METODICA DI ESTRAZIONE DEL DNA PER USO FORENSE DA REPERTI OSSEI CARBONIZZATI

Screening of subtelomeric rearrangements in autism spectrum disorder. Identification of a partial trisomy of 13q in a patient.

Analisi molecolare del gene GRPR in una popolazione autistica siciliana.

Analisi molecolare di 3 geni (NLGN3, NLGN4, GRPR) localizzati sul cromosoma X in una popolazione autistica siciliana..

Molecular analyses of NLGN3, NLGN4, GRPR genes in a Sicilian autistic population.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.