Applicazioni innovative dell’ingegneria biomedica

The recent important advances in engineering and medicine at the molecular, cellular, and tissue levels together with the significant modifications in people life style, the rapid aging, and the improved life quality expectations for the world population make urgent for the scientific committee to learn more about human body functions at a macro-, micro-, and nano-scale. In this context, Biomedical Engineering has emerged: it uses methods and techniques proper of the engineer field to describe, understand, and solve medical and biological issues and it allows the cooperation among engineers, bio-physicists, physicians, and biologists. Tissue engineering is one of the main branches of Biomedical Engineering and it has sprout to satisfy the need of designing and building living biological tissues in vitro. This is for sure a promising alternative to transplantation, especially to autologous transplantation. This strategy of growing and using autologous tissues as grafts possibly makes available tissues/organs immunologically compatible with the recipient, thus avoiding immunosuppressive therapies and partially solving the constant shortage of donor tissues and organs. Among recent strategies to obtain tissues in vitro, decellularization has developed. This method aims at producing acellular biological matrices to be used as scaffolds for new organs suitable for transplantation. The first part of PhD has dealt with the optimization of a decellularization protocol for ovine carotids, designed to obtain an adequate biological matrix for small vascular graft. Besides, progresses and growth of Biomedical Engineering imply the rapid spreading of innovative materials. As a consequence, in order to guarantee the appropriate protection of human health and of the environment, it is essential to foresee eventual adverse effects of the exposure to such new materials, and evaluate their potential risk, both with toxicological analyses and preclinical and clinical studies. In particular, this is a crucial issue within one of the most recent fields of Biomedical Engineering, i.e. nanotechnologies, which are still lacking a specific regulation. This is the reason why the second part of PhD thesis is focused on the development of a new acute oral toxicological test in vitro, based on the use of human mesenchymal stem cells (hMSCs) and designed according to ICCVAM guidelines. These were drafted during the validation process of the two in vitro tests which are now approved and used, despite their limits, to evaluate acute oral toxicology and to predict the starting dose for acute toxicity tests in vivo (ICCVAM, 2006). Considering the promising results obtained, the hMSC test was adopted to estimate nanoparticle cytotoxicity

Microbial diversity of traditional Sicilian cheeses

Traditional Sicilian cheeses are manufactured in small size farms with raw milk from animals of indigenous breeds and without the addition of starter cultures. In order to transform milk into cheese, the presence of lactic acid bacteria (LAB) is required. The main sources of desirable LAB are generally the milk, the rennet, the equipment used during processing and the dairy environment. In the last years, the microbial characterisation of traditional Sicilian cheeses, such as Caciocavallo Palermitano, Protected Designation of Origin (PDO) Pecorino Siciliano and PDO Vastedda della valle del Belìce have been the object of different studies conducted by our research group. To this purpose, the aim of the present study was to describe the microbial population of traditional Sicilian cheeses. The analysis of the microbial diversity of these cheeses revealed the presence of several dairy LAB at dominant levels, while the undesired microorganisms, including coliforms and coagulase-positive staphylococci (CPS), were at very low densities. Overall, the presence of the pathogenic bacteria Listeria monocytogenes and Salmonella spp. was never detected. This may be due to the quality of milk, the optimal maintenance of wooden vats and on following good production practices. Furthermore, the technological characterisation of the LAB found in these cheeses and in raw materials showed interesting dairy properties, including acidification capacity, diacetyl formation, autolytic properties and the ability to inhibit undesired bacteria

Oleuropein-Laded Ufasomes Improve the Nutraceutical Efficacy

Ufasomes are unsaturated fatty acid liposomes made up of oleic and linoleic acids, natural components required in various biological processes. This kind of nanocarrier is characterized by a simple and dynamic structure and is able to improve the bioavailability of unsaturated fatty acids. The aim of this investigation was to evaluate ufasomes as natural compound delivery systems to deliver oleuropein and improve its antioxidant activity. Oleuropein is a phenolic compound mainly present in olives and olive oil, with several biological properties, such as the antioxidant activity. However, to improve their biological activity, antioxidant compounds should be able to cross cell membranes and uniformly incorporate in cells. Because of the great similarity between their constituents and cell membranes, ufasomes could be advantageous carriers for oleuropein delivery. The physico-chemical characteristics of ufasomes were investigated. A regular shape was shown by transmission electron microscopy studies, while the mean sizes were dependent on the ufasomes composition. In vitro studies highlighted that empty ufasomes did not lead to cell mortality at the tested concentrations and a good carrier internalization in CaCo-2 cells, further studies in vitro studies demonstrated that oleuropein-loaded ufasomes were able to enhance the antioxidant activity of the free active substance making this carrier a suitable one for nutraceutical application

Metagenomic approach to identify the complex microbiota in Sicilian cheeses

The results obtained when identifying the microbiota in Sicilian cheeses were consistent for both taxonomic identification and abundance. Most of the genera detected in this work have often been described as a part of the microbiota of nonpasteurised milk.peer-reviewe

Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2

Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodelling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A (PKA) signalling appears to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signalling microdomains. Objective: How individual cAMP microdomains impact on cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results: Using pharmacological and genetic manipulation of PDE activity we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy whereas increasing cAMP levels via PDE2 inhibition is anti-hypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of PKA isoforms we demonstrate that the anti-hypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a PKA type II subset leading to phosphorylation of the nuclear factor of activated T cells (NFAT). Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo and its inhibition may have therapeutic applications

Microbial diversity of traditional Sicilian cheeses

In the last years, the microbial characterisation of traditional Sicilian cheeses, such as Caciocavallo Palermitano, Protected Designation of Origin (PDO) Pecorino Siciliano and PDO Vastedda della valle del Belìce have been the object of different studies conducted by our research group. To this purpose, the aim of the present study was to describe the microbial population of traditional Sicilian cheeses.peer-reviewe

Reduction of PDO Pecorino Siciliano cheese making duration: microbial dynamics and quality attributes deriving from replacing whey permeate with hot water during cooking

This work was carried out with the aim to reduce the transformation duration of Protected Designation of Origin (PDO) Pecorino Siciliano cheese. To this purpose, the cooking in hot water (experimental production, EXP) was compared to the traditional cheese cooking under whey permeate (control production, CTR). The microbiological composition of under rind (UR) and core (Co) section of CTR and EXP cheeses was determined by a combined culture-dependent and -independent approach. Total mesophilic microorganisms and lactic acid bacteria (LAB) present in raw ewes' milk (5.0 log CFU/mL) increased during cheese making and reached values of about 8.0 log CFU/g in both sections (UR and Co) of 5-month ripened cheeses of both productions (CTR and EXP) monitored. The identification of the viable LAB populations in ripened cheeses showed that Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus and Streptococcus dominated UR and Co sections of all cheeses. MiSeq Illumina analysis demonstrated that LAB populations (lactobacilli, lactococci and streptococci) dominated the bacterial community of cheeses at 95.63-98.41 % of relative abundance. The two different cooking operations did not influence the physicochemical characteristics of PDO Pecorino Siciliano cheeses. Sensory evaluation performed by artificial senses analysis and trained panelists confirmed that the modification of PDO Pecorino Siciliano cheese production protocol did not significantly affect product characteristics and overall acceptance. Thus, data of this work confirmed that cooking under hot water allowed to reduce transformation duration and safeguard typicality of PDO Pecorino Siciliano cheese

'Less is more': validation with Rasch analysis of five short-forms for the Brain Injury Rehabilitation Trust Personality Questionnaires (BIRT-PQs).

Background: Previous analyses demonstrated a lack of unidimensionality, item redundancy, and substantial administrative burden for the Brain Injury Rehabilitation Trust Personality Questionnaires (BIRT-PQs). Objective: To use Rasch Analysis to calibrate five short-forms of the BIRT-PQs, satisfying the Rasch model requirements. Methods: BIRT-PQs data from 154 patients with severe Acquired Brain Injury (s-ABI) and their caregivers (total sample = 308) underwent Rasch analysis to examine their internal construct validity and reliability according to the Rasch model. Results: The base Rasch analyses did not show sufficient internal construct validity according to the Rasch model for all five BIRT-PQs. After rescoring 18 items, and deleting 75 of 150 items, adequate internal construct validity was achieved for all five BIRT-PQs short forms (model chi-square p-values ranging from 0.0053 to 0.6675), with reliability values compatible with individual measurements. Conclusions: After extensive modifications, including a 48% reduction of the item load, we obtained five short forms of the BIRT-PQs satisfying the strict measurement requirements of the Rasch model. The ordinal-to-interval measurement conversion tables allow measuring on the same metric the perception of the neurobehavioral disability for both patients with s-ABI and their caregivers

Prediction of early recurrent thromboembolic event and major bleeding in patients with acute stroke and atrial fibrillation by a risk stratification schema: the ALESSA score study

Background and Purposes—This study was designed to derive and validate a score to predict early ischemic events and major bleedings after an acute ischemic stroke in patients with atrial fibrillation. Methods—The derivation cohort consisted of 854 patients with acute ischemic stroke and atrial fibrillation included in prospective series between January 2012 and March 2014. Older age (hazard ratio 1.06 for each additional year; 95% confidence interval, 1.00–1.11) and severe atrial enlargement (hazard ratio, 2.05; 95% confidence interval, 1.08–2.87) were predictors for ischemic outcome events (stroke, transient ischemic attack, and systemic embolism) at 90 days from acute stroke. Small lesions (≤1.5 cm) were inversely correlated with both major bleeding (hazard ratio, 0.39; P=0.03) and ischemic outcome events (hazard ratio, 0.55; 95% confidence interval, 0.30–1.00). We assigned to age ≥80 years 2 points and between 70 and 79 years 1 point; ischemic index lesion >1.5 cm, 1 point; severe atrial enlargement, 1 point (ALESSA score). A logistic regression with the receiver-operating characteristic graph procedure (C statistic) showed an area under the curve of 0.697 (0.632–0.763; P=0.0001) for ischemic outcome events and 0.585 (0.493–0.678; P=0.10) for major bleedings. Results—The validation cohort consisted of 994 patients included in prospective series between April 2014 and June 2016. Logistic regression with the receiver-operating characteristic graph procedure showed an area under the curve of 0.646 (0.529–0.763; P=0.009) for ischemic outcome events and 0.407 (0.275–0.540; P=0.14) for hemorrhagic outcome events. Conclusions—In acute stroke patients with atrial fibrillation, high ALESSA scores were associated with a high risk of ischemic events but not of major bleedings

Missense mutations in Desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro

<p>Abstract</p> <p>Background</p> <p>Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement.</p> <p>We screened 54 ARVC probands for mutations in desmocollin-2 (<it>DSC2</it>), the only desmocollin isoform expressed in cardiac tissue.</p> <p>Methods</p> <p>Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing.</p> <p>To evaluate the pathogenic potentials of the <it>DSC2 </it>mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.</p> <p>Results</p> <p>We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions.</p> <p>In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.</p> <p>Conclusion</p> <p>The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.</p