Aldo Nicosia

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37.

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended.

Role of high-resolution ultrasonography without and with real-time spatial compound imaging in evaluating the injured posterior cruciate ligament: preliminary study

PurposeThis study sought to compare high-resolution ultrasonography (HRUS) without and with compound imaging in evaluating the injured posterior cruciate ligament (PCL).Materials and methodsThirteen patients with a magnetic resonance imaging (MRI) diagnosis of PCL lesions (ten acute and three chronic) and 20 healthy controls underwent conventional and compound HRUS performed by the same radiologist who was blinded to the subjects’ case-control status. The PCL was scanned in a longitudinal direction in all cases. HRUS images were assessed for PCL thickness by two other radiologists blinded to the number and type of PCL injury. PCLs were classified as normal or injured, and the latter as having acute or chronic injuries. Intermethod reproducibility of measuring PCL thickness was assessed on conventional and compound HRUS images.ResultsComplete agreement in classifying normal or injured PCL and acute or chronic PCL injuries was achieved. PCL thickness in volunteers was 4.5±0.7 mm on conventional images and 4.6±0.7 mm on compound images. On conventional and compound HRUS images, the thickness of acutely injured PCL was 9.1±1.5 mm and 9.2±1.7 mm, respectively, and that of chronically injured PCL was 7±0.9 mm and 7±0.8 mm. Intermethod reproducibility of PCL thickness measurements on conventional vs. compound HRUS images was 98.6%.ConclusionsHRUS is a reliable technique for studying the PCL and detecting PCL injuries.AbstractObiettivoConfrontare l’ecografia ad alta risoluzione (HRUS) senza e con il compound nello studio del legamento crociato posteriore (LCP) con lesioni traumatiche.Materiali e metodiTredici pazienti con diagnosi RM di lesione del LCP, 10 acuta e 3 cronica, sono stati sottoposti ad HRUS convenzionale e compound eseguita in cieco dallo stesso radiologo. Il gruppo controllo HRUS era composto da 20 volontari sani e sono state eseguite scansioni HRUS longitudinali del LCP. In consenso ed in cieco altri due radiologi hanno misurato nelle immagini HRUS lo spessore degli LCP, quindi i casi sono stati classificati in normali/patologici e questi ultimi in acuti/cronici. È stata valutata la riproducibilità intermetodo nella misurazione dello spessore degli LCP nelle immagini convenzionali versus compound.RisultatiNella classificazione del LCP normale/patologico e con lesione acuta/cronica abbiamo evidenziato la completa concordanza HRUS/RM e HRUS convenzionale/compound. Nelle immagini HRUS convenzionali e compound lo spessore del LCP era rispettivamente nel normale 4,5±0,7 mm e 4,6±0,7 mm, nelle lesioni acute 9,1±1,5 mm e 9,2±1,7 mm e nelle croniche 7±0,9 mm e 7±0,8 mm. La riproducibilità intermetodo nella misurazione dello spessore del LCP nelle immagini convenzionali versus compound era del 98,6%.ConclusioniL’HRUS consente lo studio del LCP con riconoscimento incidentale delle alterazioni traumatiche.

High-resolution ultrasonography (HRUS) of the meniscal cyst of the knee: Our experience

PurposeThis study was undertaken to assess the diagnostic accuracy of high-resolution ultrasonography (HRUS) in the detection of meniscal cysts.Materials and methodsOver a 2-year period, 1,857 patients underwent magnetic resonance imaging (MRI) of the knee for traumatic or degenerative disorders. All patients with MRI evidence of a meniscal cyst were studied by HRUS. HRUS was also performed on an equal number of patients without MRI evidence of meniscal cyst who were used as a control group. All HRUS examinations were conducted by a radiologist blinded to the MRI findings. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRUS were assessed with reference to MRI. All patients underwent surgery, and the resected masses were studied by histological examination.ResultsMRI allowed identification of a meniscal cyst in 52 patients. HRUS enabled correct detection of the meniscal cyst in 49/52 cases. In the control group, HRUS excluded the presence of meniscal cysts in all cases. HRUS had a sensitivity, specificity, PPV and NPV of 94.23%, 100%, 100% and 94.54%, respectively, for the detection of meniscal cysts.ConclusionsHRUS is a fairly reliable technique in the detection, characterisation and differentiation of the different forms of meniscal cyst.RiassuntoObiettivoVerificare l’accuratezza diagnostica dell’ecografia ad elevata risoluzione (HRUS) nella detezione delle cisti parameniscali.Materiali e metodiIn un periodo di 2 anni, sono stati sottoposti ad RM del ginocchio per patologia traumatica o degenerativa 1857 pazienti. Tutti i pazienti in cui è stata identificata una cisti parameniscale sono stati sottoposti ad HRUS. Come gruppo controllo sono stati reclutati altrettanti pazienti, esenti da cisti parameniscali. Tutte le indagini HRUS sono state eseguite in cieco da un’altro radiologo. Sono state valutate la sensibilità la specificità, il valore predittivo positivo (VPP) e negativo (VPN) dell’HRUS in comparazione con la RM. Tutti i pazienti sono stati sottoposti ad intervento chirurgico e le formazioni resecate ad esame istologico.RisultatiIn 52 pazienti l’indagine RM ha consentito l’identificazione di una cisti parameniscale. L’HRUS ne ha consentito la detezione in 49/52 casi. Nel gruppo controllo l’HRUS ne ha escluso la presenza in tutti i casi. L’HRUS ha manifestato nella detezione delle cisti parameniscali sensibilità, specificità, VPP e VPN del 94,23%, 100%, 100% e 94,54%, rispettivamente. L’intervento chirurgico e l’istologia hanno confermato la diagnosi RM.ConclusioniL’HRUS è una metodica abbastanza affidabile nell’identificazione, nella valutazione delle caratteristiche semeiologiche e nella distinzione delle differenti forme delle cisti meniscali.

Complete decontamination and regeneration of DNA purification silica columns

Silica columns are among the most used DNA purification systems, allowing a good yield of high-quality nucleic acids without organic extractions. Silica column regeneration protocols reported up to now to remove DNA traces are time-consuming, and their effectiveness on genomic DNA has not been demonstrated. Here we report a very rapid regeneration procedure that ensures no DNA carryover, independent of its size, without impairing column efficiency. The method takes advantage of the improved DNA removal by low concentrations of Triton X-100.

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257xa0bp with a 5′-untranslated region (UTR) of 63xa0bp and a 3′-UTR of 331xa0bp with a poly (A) tail, and an open-reading frame (ORF) of 864xa0bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33xa0kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.

A novel putative interactor for the low density lipoprotein receptor cytoplasmic domain.

Familial hypercholesterolemia (FH) is a genetic disease mainly caused by mutations in the low density lipoprotein receptor (LDL-R) gene. However, FH-like phenotypes may also arise from mutations occurring in other genes, the products of which normally interact with the LDL receptor. Although several FH-associated proteins have been discovered, many FH-like phenotypes cannot be linked to mutations in already characterized genes, suggesting the existence of other genes still to be identified, the mutations of which may be directly linked to the FH disorder. In order to identify new putative LDLr interactors possibly involved in its internalization and/or sorting, the cytoplasmic tail of the receptor was used as bait in a two-hybrid assay. We identified an 85-amino acid protein able to bind the LDLr intracellular domain through the last 14 C-terminal amino acids. The novel protein is probably derived from the translation of an alternative open reading frame of the human MT2A gene.