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The stromal vascular fraction (SVF) is a heterogeneous population of stem/stromal cells isolated from perivascular and extracellular matrix (ECM) of adipose tissue complex (ATC). Administration of SVF holds a strong therapeutic potential for regenerative and wound healing medicine applications aimed at functional restoration of tissues damaged by injuries or chronic diseases. SVF is commonly divided into cellular stromal vascular fraction (cSVF) and tissue stromal vascular fraction (tSVF). Cellular SVF is obtained from ATC by collagenase digestion, incubation/isolation, and pelletized by centrifugation. Enzymatic disaggregation may alter the relevant biological characteristics of adipose tissue, while providing release of complex, multiattachment of cell-to-cell and cell-to-matrix, effectively eliminating the bioactive ECM and periadventitial attachments. In many countries, the isolation of cellular elements is considered as a "more than minimal" manipulation, and is most often limited to controlled clinical trials and subject to regulatory review. Several alternative, nonenzymatic methods of adipose tissue processing have been developed to obtain via minimal mechanical manipulation an autologous tSVF product intended for delivery, reducing the procedure duration, lowering production costs, decreasing regulatory burden, and shortening the translation into the clinical setting. Ideally, these procedures might allow for the integration of harvesting and processing of adipose tissue for ease of injection, in a single procedure utilizing a nonexpanded cellular product at the point of care, while permitting intraoperative autologous cellular and tissue-based therapies. Here, we review and discuss the options, advantages, and limitations of the major strategies alternative to enzymatic processing currently developed for minimal manipulation of adipose tissue. Stem Cells Translational Medicine 2019;8:1265&1271., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Centrosomal p53 has been described for three decades but its role is still unclear. We previously reported that, in proliferating human cells, p53 transiently moves to centrosomes at each mitosis. Such p53 mitotic centrosome localization (p53-MCL) occurs independently from DNA damage but requires ATM-mediated p53Ser15 phosphorylation (p53Ser15 P ) on discrete cytoplasmic p53 foci that, through MT dynamics, move to centrosomes during the mitotic spindle formation. Here, we show that inhibition of p53-MCL, obtained by p53 depletion or selective impairment of p53 centrosomal localization, induces centrosome fragmentation in human nontransformed cells. In contrast, tumor cells or mouse cells tolerate p53 depletion, as expected, and p53-MCL inhibition. Such tumor- and species-specific behavior of centrosomal p53 resembles that of the recently identified sensor of centrosome-loss, whose activation triggers the mitotic surveillance pathway in human nontransformed cells but not in tumor cells or mouse cells. The mitotic surveillance pathway prevents the growth of human cells with increased chance of making mitotic errors and accumulating numeral chromosome defects. Thus, we evaluated whether p53-MCL could work as a centrosome-loss sensor and contribute to the activation of the mitotic surveillance pathway. We provide evidence that centrosome-loss triggered by PLK4 inhibition makes p53 orphan of its mitotic dock and promotes accumulation of discrete p53Ser15 P foci. These p53 foci are required for the recruitment of 53BP1, a key effector of the mitotic surveillance pathway. Consistently, cells from patients with constitutive impairment of p53-MCL, such as ATM- and PCNT-mutant carriers, accumulate numeral chromosome defects. These findings indicate that, in nontransformed human cells, centrosomal p53 contributes to safeguard genome integrity by working as sensor for the mitotic surveillance pathway.

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM (ox) IRPPY peptide (monoisotopic [M + 2 H] +2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called 'Lovers of Modena', two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the 'Lovers of Modena' (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or anti-DSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2 -/- immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus.

High affinity copper binding to mitogen-activated protein kinase kinase 1 (MAP2K1, also known as MEK1) allosterically promotes the kinase activity of MEK1/2 on extracellular signal regulated kinases 1 and 2 (ERK1/2). Consequently, copper-dependent activation of the mitogen-activated (MAP) kinase pathway has a role in promoting tumor growth. Conversely, copper chelation may represent a possible therapeutic approach for a specific subset of tumors characterized by activating mutations in the serine/threonine protein kinase V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF), such as the V600E, occurring within the kinase domain (BRAF V600E ). Tetrathiomolybdate (TM) is a specific copper chelating agent currently used for the treatment of Wilson's disease and in preclinical studies for the management of metastatic cancers owing to its anti-angiogenic and anti-inflammatory properties. We evaluated in vitro and in vivo the effects of copper depletion achieved by pharmacological treatment with TM in human colorectal cells bearing the BRAF V600E mutation in comparison with BRAF wild type cells. We provide evidence that selective copper chelation differentially affects proliferation, survival and migration of colon cancer cells bearing the BRAF V600E mutation compared to BRAF wt acting via differential phosphorylation levels of ERK1/2. Moreover, tetrathiomolybdate treatment was also effective in reducing the clonogenic potential of colon cancer BRAF V600E cells resistant to BRAF pharmacological inhibition. In conclusion, these results support further assessment of copper chelation therapy as an adjuvant therapy for inhibiting the progression of colon cancers containing the BRAF V600E mutation.

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O 2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O 2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( K d ) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the K i values were 3.6 ± 0.3 nM (IC 50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC 50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

Stem cells drive embryonic and fetal development. In several adult tissues, they retain the ability to self-renew and differentiate into a variety of specialized cells, thus contributing to tissue homeostasis and repair throughout life span. Alcohol consumption is associated with an increased risk for several diseases and conditions. Growing and developing tissues are particularly vulnerable to alcohol's influence, suggesting that stem- and progenitor-cell function could be affected. Accordingly, recent studies have revealed the possible relevance of alcohol exposure in impairing stem-cell properties, consequently affecting organ development and injury response in different tissues. Here, we review the main studies describing the effects of alcohol on different types of progenitor/stem cells including neuronal, hepatic, intestinal and adventitial progenitor cells, bone-marrow-derived stromal cell, dental pulp, embryonic and hematopoietic stem cells, and tumor-initiating cells. A better understanding of the nature of the cellular damage induced by chronic and episodic heavy (binge) drinking is critical for the improvement of current therapeutic strategies designed to treat patients suffering from alcohol-related disorders.

It has been well established that moderate alcohol consumption inversely correlates with cardiovascular morbidity and mortality, whereas binge alcohol drinking increases cardiovascular disease risk. The aim of this study was to assess in vivo the impact of different drinking patterns on reverse cholesterol transport (RCT); the atheroprotective process leading to the removal of excess cholesterol from the body. RCT was measured with a standardized, radioisotope-based technique in three groups of atherosclerosis-prone apolipoprotein E knock out mice: Placebo group, receiving water, which would mimic the abstainers; moderate group, receiving 0.8 g/kg alcohol/day for 28 days, which would mimic a moderate intake; binge group, receiving 0.8 g/kg alcohol/day for 5 days/week, followed by the administration of 2.8 g/kg alcohol/day for 2 days/week, which would mimic a heavy intake in a short period. Mice in the binge drinking group displayed an increase in total cholesterol, high density lipoprotein cholesterol (HDL-c) and non-HDL-c (all p < 0.0001 vs. placebo), and a significantly reduced elimination of fecal cholesterol. The moderate consumption did not lead to any changes in circulating lipids, but slightly improved cholesterol mobilization along the RCT pathway. Overall, our data confirm the importance of considering not only the total amount, but also the different consumption patterns to define the impact of alcohol on cardiovascular risk.

Background: Cell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia., Methods: We achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease., Results: We observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment., Conclusions: Collectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties.

Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using UV-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the SS bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (ΔH°' rc ) and entropy (ΔS°' rc ), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations., Competing Interests: The authors declare no conflict of interest.

The sirtuin family comprises seven NAD + -dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins' levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins' expression and clinical and molecular parameters were also analyzed. We found that sirtuin1-6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1-6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins' expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins' levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral adipose-derived stem cells of obese patients.

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

HIPK2 is a Y-regulated S/T kinase involved in various cellular processes, including cell-fate decision during development and DNA damage response. Cis-autophosphorylation in the activation-loop and trans-autophosphorylation at several S/T sites along the protein are required for HIPK2 activation, subcellular localization, and subsequent posttranslational modifications. The specific function of a few of these autophosphorylations has been recently clarified; however, most of the sites found phosphorylated by mass spectrometry in human and/or mouse HIPK2 are still uncharacterized. In the process of studying HIPK2 in human colorectal cancers, we identified a mutation (T566P) in a site we previously found autophosphorylated in mouse Hipk2. Biochemical and functional characterization of this site showed that compared to wild type (wt) HIPK2, HIPK2-T566P maintains nuclear-speckle localization and has only a mild reduction in kinase and growth arresting activities upon overexpression. Next, we assessed cell response following UV-irradiation or treatment with doxorubicin, two well-known HIPK2 activators, by evaluating cell number and viability, p53-Ser46 phosphorylation, p21 induction, and caspase cleavage. Interestingly, cells expressing HIPK2-T566P mutant did not respond to UV-irradiation, while behaved similarly to wt HIPK2 upon doxorubicin-treatment. Evaluation of HIPK2-T566 phosphorylation status by a T566-phospho-specific antibody showed constitutive phosphorylation in unstressed cells, which was maintained after doxorubicin-treatment but inhibited by UV-irradiation. Taken together, these data show that HIPK2-T566 phosphorylation contributes to UV-induced HIPK2 activity but it is dispensable for doxorubicin response.

The correct staging of disease, with an exact definition of the extent of cancer at the diagnosis, is crucial in the planning of a specific treatment and in the assessment of real chances of cure. Cancer staging systems are expected to be accurate in the description of the severity of a patient's tumor on the basis of the extent of the primary neoplasm and of its spread, thus giving clinician tools to estimate prognosis and providing objective parameters to compare groups of patients in clinical studies. This last point is of wide importance in evaluating successful treatment strategies in oncology, and this is one of the issues that contributed to the development of stage-adapted therapies., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction., Competing Interests: The authors declare no conflict of interest.

The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

Seventh tumor-node-metastasis (TNM) classification for gastric cancer, published in 2010, introduced changes in all of its three parameters with the aim to increase its accuracy in prognostication. The aim of this review is to analyze the efficacy of these changes and their implication in clinical practice. We reviewed relevant Literature concerning staging systems in gastric cancer from 2010 up to March 2016. Adenocarcinoma of the esophago-gastric junction still remains a debated entity, due to its peculiar anatomical and histological situation: further improvement in its staging are required. Concerning distant metastases, positive peritoneal cytology has been adopted as a criterion to define metastatic disease: however, its search in clinical practice is still far from being routinely performed, as staging laparoscopy has not yet reached wide diffusion. Regarding definition of T and N: in the era of multimodal treatment these parameters should more influence both staging and surgery. The changes about T-staging suggested some modifications in clinical practice. Differently, many controversies on lymph node staging are still ongoing, with the proposal of alternative classification systems in order to minimize the extent of lymphadenectomy. The next TNM classification should take into account all of these aspects to improve its accuracy and the comparability of prognosis in patients from both Eastern and Western world., Competing Interests: Conflict-of-interest statement: None to declare.

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Objectives: To assess whether ultrasound elastography (USE) with strain ratio increases diagnostic accuracy of Doppler ultrasound in further characterisation of cytologically Thy3 thyroid nodules., Methods: In two different university diagnostic centres, 315 patients with indeterminate cytology (Thy3) in thyroid nodules aspirates were prospectively evaluated with Doppler ultrasound and strain ratio USE before surgery. Ultrasonographic features were analysed separately and together as ultrasound score, to assess sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Receiver operating characteristic (ROC) curves to identify optimal cut-off value of the strain ratio were also provided. Diagnosis on a surgical specimen was considered the standard of reference., Results: Higher strain ratio values were found in malignant nodules, with an optimum strain ratio cut-off of 2.09 at ROC analysis. USE with strain ratio showed 90.6% sensitivity, 93% specificity, 82.8% PPV, 96.4% NPV, while US score yielded a sensitivity of 52.9%, specificity of 84.3%, PPV 55.6% and NPV 82.9%. The diagnostic gain with strain ratio was statistically significant as proved by ROC areas, which was 0.9182 for strain ratio and 0.6864 for US score., Conclusions: USE with strain ratio should be considered a useful additional tool to colour-Doppler US, since it improves characterisation of thyroid nodules with indeterminate cytology., Key Points: • Strain ratio measurements improve differentiation of thyroid nodules with indeterminate cytology • Elastography with strain ratio is more reliable than ultrasound features and ultrasound score • Strain ratio may help to better select patients with Thy 3 nodules candidate for surgery.

Competing Interests: The authors have no conflicts of interest to declare.

Background: Granzyme B (GrB) is a serine protease, traditionally known as expressed by cytotoxic lymphocytes to induce target cell apoptosis. However, it is emerging that GrB, being also produced by a variety of normal and neoplastic cells and potentially acting on multiple targets, might represent a powerful regulator of a wide range of fundamental biological processes. We have previously shown that GrB is expressed in urothelial carcinoma tissues and its expression is associated to both pathological tumor spreading and EMT. We have also shown that docosahexaenoic acid (DHA), a dietary ω-3 polyunsaturated fatty acid with anti-tumor activity, while inhibiting urothelial and pancreatic carcinoma cell invasion also inhibited their GrB expression in vitro. In this study, we characterized a panel of colorectal carcinoma (CRC) cells, with different invasive capabilities, for GrB expression and for the contribution of GrB to their EMT and invasive phenotype. In addition, we investigated the effect of DHA on CRC cell-associated GrB expression, EMT and invasion., Methods: The expression levels of GrB and EMT-related markers were evaluated by Western blotting. GrB knockdown was performed by Stealth RNAi small interfering RNA silencing and ectopic GrB expression by transfection of human GrB vector. Cell invasion was determined by the BioCoat Matrigel invasion chamber test., Results: GrB was produced in 57.1% CRC cell lines and 100% CRC-derived Cancer Stem Cells. Although GrB was constitutive expressed in both invasive and noninvasive CRC cells, GrB depletion in invasive CRC cells downmodulated their invasion in vitro, suggesting a contribution of GrB to CRC invasiveness. GrB loss or gain of function downmodulated or upmodulated EMT, respectively, according to the analysis of cancer cell expression of three EMT biomarkers (Snail1, E-cadherin, N-cadherin). Moreover, TGF-β1-driven EMT was associated to the enhancement of GrB expression in CRC cell lines, and GrB depletion led to downmodulation of TGF-β1-driven EMT. In addition, DHA inhibited GrB expression, EMT and invasion in CRC cells in vitro., Conclusions: These findings present a novel role for GrB as upmodulator of EMT in CRC cells. Moreover, these results support the use of DHA, a dietary compound without toxic effects, as adjuvant in CRC therapy.

Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects.

Extracellular vesicles (EVs), such as microvesicles and exosomes, are membranous structures containing bioactive material released by several cells types, including mesenchymal stem/stromal cells (MSCs). Increasing lines of evidences point to EVs as paracrine mediators of the beneficial effects on tissue remodeling associated with cell therapy. Administration of MSCs-derived EVs has therefore the potential to open new and safer therapeutic avenues, alternative to cell-based approaches, for degenerative diseases. However, an enhanced knowledge about in vivo EVs trafficking upon delivery is required before effective clinical translation. Only a few studies have focused on the biodistribution analysis of exogenously administered MSCs-derived EVs. Nevertheless, current strategies for in vivo tracking in animal models have provided valuable insights on the biodistribution upon systemic delivery of EVs isolated from several cellular sources, indicating in liver, spleen, and lungs the preferential target organs. Different strategies for targeting EVs to specific tissues to enhance their therapeutic efficacy and reduce possible off-target effects have been investigated. Here, in the context of a possible clinical application of MSC-derived EVs for tissue regeneration, we review the existing strategies for in vivo tracking and targeting of EVs isolated from different cellular sources and the studies elucidating the biodistribution of exogenously administered EVs., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Background: Total thyroidectomy (TT) and subtotal thyroidectomy (ST) are worldwide treatment options for multinodular non-toxic goitre in adults. Near TT, defined as a postoperative thyroid remnant less than 1 mL, is supposed to be a similarly effective but safer option than TT. ST has been shown to be marginally safer than TT, but it may leave an undetected thyroid cancer in place., Objectives: The objective was to assess the effects of total or near-total thyroidectomy compared to subtotal thyroidectomy for multinodular non-toxic goitre., Search Methods: We searched the Cochrane Library, MEDLINE, PubMed, EMBASE, as well as the ICTRP Search Portal and ClinicalTrials.gov. The date of the last search was 18 June 2015 for all databases. No language restrictions were applied., Selection Criteria: Two review authors independently scanned the abstract, title or both sections of every record retrieved to identify randomised controlled trials (RCTs) on thyroidectomy for multinodular non-toxic goitre for further assessment., Data Collection and Analysis: Two review authors independently extracted data, assessed studies for risk of bias and evaluated overall study quality utilising the GRADE instrument. We calculated the odds ratio (OR) and corresponding 95% confidence interval (CI) for dichotomous outcomes. A random-effects model was used for pooling data., Main Results: We examined 1430 records, scrutinized 14 full-text publications and included four RCTs. Altogether 1305 participants entered the four trials, 543 participants were randomised to TT and 762 participants to ST. A total of 98% and 97% of participants finished the trials in the TT and ST groups, respectively. Two trials had a duration of follow-up between 12 and 39 months and two trials a follow-up of 5 and 10 years, respectively. Risk of bias across studies was mainly unknown for selection, performance and detection bias. Attrition bias was generally low and reporting bias high for some outcomes. In the short-term postoperative period no deaths were reported for both TT and ST groups. However, longer-term data on all-cause mortality were not reported (1284 participants; 4 trials; moderate quality evidence). Goiter recurrence was lower in the TT group compared to ST. Goiters recurred in 0.2% (1/425) of the TT group compared to 8.4% (53/632) of the ST group (OR 0.05 (95% CI 0.01 to 0.21); P < 0.0001; 1057 participants; 3 trials; moderate quality evidence). Re-intervention due to goitre recurrence was lower in the TT group compared to ST. Re-intervention was necessary in 0.5% (1/191) of TT patients compared to 0.8% (3/379)of ST patients (OR 0.66 (95% CI 0.07 to 6.38); P = 0.72; 570 participants; 1 trial; low quality evidence). The incidence of permanent recurrent laryngeal nerve palsy was lower for ST compared with TT. Permanent recurrent laryngeal nerve palsy occurred in 0.8% (6/741) of ST patients compared to 0.7% (4/543) of TT patients (OR 1.28, (95% CI 0.38 to 4.36); P = 0.69; 1275 participants; 4 trials; low quality evidence). The incidence of permanent hypoparathyroidism was lower for ST compared with TT. Permanent hypoparathyroidism occurred in 0.1% (1/741) of ST patients compared to 0.6% (3/543) of TT patients (OR 3.09 (95% CI 0.45 to 21.36); P = 0.25; 1275 participants: 4 trials; low quality evidence). The incidence of thyroid cancer was lower for ST compared with TT. Thyroid cancer occurred in 6.1% (41/669) of ST patients compared to 7.3% (34/465)of TT patients (OR 1.32 (95% CI 0.81 to 2.15); P = 0.27; 1134 participants; 3 trials; low quality evidence). No data on health-related quality of life or socioeconomic effects were reported in the included studies., Authors' Conclusions: The body of evidence on TT compared with ST is limited. Goiter recurrence is reduced following TT. The effects on other key outcomes such as re-interventions due to goitre recurrence, adverse events and thyroid cancer incidence are uncertain. New long-term RCTs with additional data such as surgeons level of experience, treatment volume of surgical centres and details on techniques used are needed.

Background: Obesity is a major risk factor for several chronic diseases. The BioEnterics(®) Intragastric Balloon (BIB(®)) is used worldwide as a short-term (6 months) treatment in morbid obese patients. However, removal of BIB(®) past 6 months is a common occurrence in clinical practice often as a result of patient factors. The aim of the present Italian multicentre cohort study was to evaluate the safety and efficacy of the BIB(®) left in situ for more than 6 months., Materials and Methods: A total of eight Italian centres participated in the study. Participating centres received a standardised questionnaire designed to capture safety and efficacy data. Weight loss data, as well as date, and reason for removal of the BIB(®) after 6 months were recorded. Adverse events, including mortality and complications, operators' subjective technical notes and findings, and difficulties during removal procedure were also collected., Results: Six hundred and eleven patients were included. In the majority of cases, patient extended BIB(®) treatment due to satisfactory weight loss (44 %). At 6 months, all patients achieved a BMI statistically lower than the initial BMI (p < 0.001). There was a non-significant trend towards greater BMIL % in patients who underwent removal up to 15 months versus the results achieved up to 6 months. No major complications were recorded., Conclusion: Extending the duration of BIB(®) use up to 14 months safely maintains weight loss and satiety with greater results than that up to 6 months, without complications.

Background: The rising interest towards minimally invasive surgery has led to the introduction of laparo-endoscopic single site (LESS) surgery as the natural evolution of conventional multiport laparoscopy. However, this new surgical approach is hampered with peculiar technical difficulties. The SPIDER surgical system has been developed in the attempt to overcome some of these challenges. Our study aimed to compare standard laparoscopy and SPIDER technical performance on a surgical simulator, using standardized tasks from the Fundamentals of Laparoscopic Surgery (FLS)., Methods: Twenty participants were divided into two groups based on their surgical laparoscopic experience: 10 PGY1 residents were included in the inexperienced group and 10 laparoscopists in the experienced group. Participants performed the FLS pegboard transfers task and pattern cutting task on a laparoscopic box trainer. Objective task scores and subjective questionnaire rating scales were used to compare conventional laparoscopy and SPIDER surgical system., Results: Both groups performed significantly better in the FLS scores on the standard laparoscopic simulator compared to the SPIDER. Inexperienced group: Task 1 scores (median 252.5 vs. 228.5; p = 0.007); Task 2 scores (median 270.5 vs. 219.0; p = 0.005). Experienced group: Task 1 scores (median 411.5 vs. 309.5; p = 0.005); Task 2 scores (median 418.0 vs. 331.5; p = 0.007). Same aspects were highlighted for the subjective evaluations, except for the inexperienced surgeons who found both devices equivalent in terms of ease of use only in the peg transfer task., Conclusions: Even though the SPIDER is an innovative and promising device, our study proved that it is more challenging than conventional laparoscopy in a population with different degrees of surgical experience. We presume that a possible way to overcome such challenges could be the development of tailored training programs through simulation methods. This may represent an effective way to deliver training, achieve mastery and skills and prepare surgeons for their future clinical experience.

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc-CL) immobilized at IMM. In this work, we have studied electrochemically cytc-CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc-CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc-CL in solution. In particular, immobilized cytc-CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/- coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH(-) states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc-CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

Purpose: To assess the best technique and the diagnostic accuracy of Quasistatic Ultrasound Elastography (QUE) in thyroid nodules. Interobserver agreement was also evaluated., Materials and Methods: A preliminary study of 50 patients with 54 thyroid nodules was performed with quantitative software in order to define the best cut-off value of different imaging methods. All patients underwent total thyroidectomy and histopathology findings served as the standard of reference. Thereafter, 154 nodules in 137 consecutive patients were prospectively evaluated by three operators. Findings at fine-needle aspiration cytology and histopathology (N = 60) served as the standard of reference., Results: The most accurate technique was the axial peri-intranodular measurement method which achieved an area under the ROC curve of 0.961 (95 %CI 0.848 - 1.00) and had an optimal cut-off value of 3.00. QUE in the differentiation of thyroid nodules showed for operator 1: sensitivity 90 % (95 %CI 73.5 - 97.9 %), specificity 92.7 % (95 %CI 86.7 - 96.6 %), LR+ 12.40 (6.54 - 23.50), LR- 0.11 (0.04 - 0.32) and accuracy 91.4 % (95 %CI 85.4 - 97.3 %); for operator 2: sensitivity 86.7 % (95 %CI 69.3 - 96.2 %), specificity 87.1 % (95 %CI 79.9 - 92.4 %), LR+ 6.72 (4.16 - 10.80), LR- 0.15 (0.06 - 0.38) and accuracy 86.9 % (95 %CI 80.0 - 93.7 %); for operator 3: sensitivity 80 % (95 %CI 61.4 - 92.3 %), specificity 83.9 % (95 %CI 76.2 - 89.9 %), LR+ 4.96 (3.20 - 7.70), LR- 0.24 (0.12 - 0.49) and accuracy 81.9 % (95 %CI 74.0 - 89.9 %). Interobserver agreement values between operator 1 and operator 2 (k = 0.79) (p < 0.05, 95 %CI 0.684 - 0.904), between operator 1 and operator 3 (k = 0.73, 95 %CI: 0.607 - 0.854) and between operator 2 and operator 3 (k = 0.71, 95 %CI: 0.584 - 0.835) were significant., Conclusion: QUE provides accurate quantitative evaluation of thyroid nodules with low interobserver variability., (© Georg Thieme Verlag KG Stuttgart · New York.)

Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (Rfact of 13.8% and Rfree of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds.

Background: Distal pancreatectomy (DP) is a surgical procedure performed to remove the pancreatic tail jointly with a variable part of the pancreatic body and including a spleen resection in the case of conventional distal pancreatectomy or not in the spleen-preserving distal pancreatectomy., Methods: In this article, we describe a standardized operative technique for fully robotic distal pancreatectomy., Results: In the last decade, the use of robotic systems has become increasingly common as an approach for benign and malignant pancreatic disease treatment. Robotic Distal Pancreatectomy (RDP) is an emerging technology for which sufficient data to draw definitive conclusions in surgical oncology are still not available because the follow-up period after surgery is too short (less than 2 years)., Conclusions: RDP is an emerging technology for which sufficient data to draw definitive conclusions of value in surgical oncology are still not available, however this techniques is safe and reproducible by surgeons that possess adequate skills.

Epigenetic control of gene expression plays a major influence in the development and progression of many cancer types. Aim of the present study was to investigate the expression of epigenetic regulators in a large cohort of medullary thyroid carcinomas (MTC), correlating the data with the clinical outcome and mutational status of the patients. Taqman Low Density Arrays (TLDAs) were used to analyze expression levels of several genes involved in the epigenetic control of transcription in a series of 54 MTCs. The patients cohort included 13 familial MTCs and 41 sporadic forms; 33 hosted a RET mutation and 13 a RAS somatic mutation. The expression profiling revealed in the more aggressive diseases (i.e. occurrence of metastases; persistent disease; disease-related death) a significant increase of EZH2 and SMYD3 gene expression. The increased levels of EZH2 and SMYD3 did not correlate significantly with mutational status of RET or RAS genes. Thus, the histone methyltransferases EZH2 and SMYD3 mRNA expression may represent useful prognostic biomarkers tailoring the most appropriate follow-up and timing of therapeutic approaches., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The K72A/K73H/K79A variant of cytochrome c undergoes a reversible change from a His/Met to a His/His axial heme ligation upon urea-induced unfolding slightly below neutral pH. The unfolded form displays a dramatically lower reduction potential than the folded species along with a pseudo-peroxidase activity. We have studied electrochemically the effects of urea-induced unfolding on the protein electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter mimics the electrostatic interaction with the inner mitochondrial membrane. This behavior has been compared with the unfolding of the same species in solution. This system constitutes a model to decipher the role of the above electrostatic interaction in the unfolding of cytochrome c at physiological pH upon interaction with the membrane component phospholipid cardiolipin in the early stages of the apoptosis cascade. We found that immobilization obstacles protein unfolding due to structural constraints at the interface imposed by protein-SAM interaction.

Thyroid pathology including thyroid nodules and diffuse thyroid diseases represents often a diagnosing challenge for clinicians. US, although highly accurate in identifying thyroid nodules and diffuse thyroid diseases, is still not sufficiently accurate to evaluate them. US-elastography has been introduced in order to further increase US accuracy in many fields and eventually for thyroid disease. The aim of the present paper it to provide an update of the literature on different available techniques and the results reported both for thyroid nodules differentiation and for diffuse thyroid disease evaluation. Advantages and limitations of elastography are also discussed., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

Purpose: The aim of this study is to assess the diagnostic efficacy and interobserver agreement of acoustic radiation force impulse (ARFI) elasticity imaging in differentiating thyroid nodules., Methods: In our study, 74 consecutive patients (52 females, 22 males; age range 27-77 years, mean: 41 years) with 82 thyroid nodules (60 benign nodules, and 22 malignant) were examined by two radiologists with different experience. Patients underwent either cytology using fine needle aspiration cytology or thyroid surgery. The diagnostic performance of the two operators at ARFI with sensitivity, specificity, positive predictive and negative predictive value, and ROC curves was estimated. Inter-reader variability between the two operators was defined using Cohen's k., Results: According to receiver operating characteristics ROC curves (AUROC = 0.86 for observer 1; 0.81 for observer 2) sensitivity, specificity, PPV and NPV of reader 1 and 2 were respectively: 90, 75, 90.91 and 96.55 %; (cut-off value of shear wave: 2.455 m/s); 90, 72, 90 and 96.90 % (cut-off value shear wave: 2.365 m/s). Concordance between the two operators was good (k = 0.755)., Conclusions: This work is a feasibility study evaluating ARFI imaging. Its results suggest that ARFI imaging is a reproducible method which can be utilized with good diagnostic performance in the thyroid for discriminating benign and malignant nodules using the cut-off value of 2.455 m/s. However, larger studies are needed to validate this method.

Wanting to find a way of identifying patients suitable for early discharge after thyroidectomy, we set out to establish whether ionized calcium concentration is a better predictor of post-surgical hypocalcemia than total serum calcium. Data were analyzed to establish whether serum ionized calcium concentrations are correlated with total serum calcium levels and symptomatic hypocalcemia after thyroidectomy. Sixty-two patients undergoing total thyroidectomy at the Department of Surgical Sciences of the "Sapienza" University of Rome, Italy, in 2010. Ionized calcium was measured before (day 0) and after surgery (days 1, 2 and 60) in all the patients. These measurements were compared with preoperative (day 0) and postoperative total serum calcium levels (days 1, 2 and 60). The preoperative ionized calcium levels differed from the ionized calcium levels recorded on days 1 and 2; this pattern was not observed for the total calcium concentrations. Conversely, total calcium on days I and II correlated significantly with the various ionized calcium measurements. The presence of parathyroid glands in the surgical specimen did not seem to affect suitability for discharge. The statistical analysis showed that ionized calcium measurements are more reliable than total calcium measurements in the immediate and long-term follow-up of total thyroidectomy patients. Applying a 95% confidence interval we established reference values for both total serum calcium and ionized calcium, below which all patients develop postoperative symptomatic hypocalcemia. In conclusion, measurement of ionized calcium, as opposed to total calcium, should be strongly recommended in the immediate and longterm follow-up of total thyroidectomy patients.

Each year, ~988,000 new cases of stomach cancer are reported worldwide. Uniformity for the definition of advanced gastric cancer (AGC) is required to ensure the improved management of patients. Various classifications do actually exist for gastric cancer, but the classification determined by lesion depth is extremely important, as it has been shown to correlate with patient prognosis; for example, early gastric cancer (EGC) has a favourable prognosis when compared with AGC. In the literature, the definition of EGC is clear, however, there is heterogeneity in the definition of AGC. In the current study, all parameters of the TNM classification for AGC reported in each previous study were individually analysed. It was necessary to perform a comprehensive systematic literature search of all previous studies that have reported a definition of ACG to guarantee homogeneity in the assessment of surgical outcome. It must be understood that the term 'advanced gastric cancer' may implicate a number of stages of disease, and studies must highlight the exact clinical TNM stages used for evaluation of the study.

Objective: Evaluation of the diagnostic efficacy and interobserver agreement of Q-elastography in the differentiation of benign from malignant thyroid nodules., Methods: A total of 344 thyroid nodules in 288 patients were examined with grey-scale and colour Doppler ultrasound (CDUS) and Q-elastography by two independent operators. Nodules with hypoechogenicity, poorly defined margins, microcalcifications, and intralesional vascularity were classified as suspicious. Diagnostic performances of CDUS features and Q-elastography for predicting thyroid malignancy were estimated using ROC analysis. Cytology or histopathology was the reference standard. Interobserver agreement in the evaluation of CDUS and Q-elastography was assessed using Cohen's k-statistic., Results: Q-elastography showed excellent diagnostic performance for the prediction of thyroid malignancy, with sensitivity of 93 % and specificity of 92 % for operator 1 (best cutoff at 2.02), and sensitivity of 84 % and specificity of 79 % for operator 2 (best cutoff at 1.86). Performance of Q-elastography was superior to that of CDUS. Reproducibility of the findings was excellent for both Q-elastography and CDUS features as assessed with Cohen's k, which was highest for strain ratio measurements (0.95) and lowest for the echogenicity score (0.83)., Conclusions: Q-elastography showed excellent performance. It is a valid and reproducible diagnostic method as well as a promising tool for identifying suspicious solid thyroid nodules needing cytological assessment and surgery., Key Points: • Elastography is an additional tool for optimal characterisation of malignant thyroid nodules. • The use of semiquantitative elastographic evaluation increases the diagnostic performance, • The interobserver agreement of quantitative elastography can be considered to be good.

Background: Construct validity of virtual laparoscopic simulators for basic laparoscopic skills has been proposed; however, it is not yet clear whether the simulators can identify the actual experience of surgeons in more complex procedures such as laparoscopic Roux-en-Y gastric bypass. This study tested the ability of the Lap Mentor simulator to recognize the experience in advanced laparoscopic procedures and to assess its role in the certification of bariatric surgeons., Methods: Twenty surgeons were divided into two groups according to their experience in laparoscopic and bariatric surgery. The general group included 10 general surgeons performing between 75 and 100 nonbariatric laparoscopic procedures. The bariatric group included 10 bariatric surgeons performing between 50 and 100 laparoscopic bariatric procedures. Participants were tested on the simulator in one basic task (task 1: eye-hand coordination) and in two tasks of the gastric bypass module (task 2: creation of the gastric pouch; task 3: gastrojejunal anastomosis)., Results: Comparing the groups, no significant differences were found in task 1. Analyzing the results from the gastric bypass module (bariatric vs. general), in task 2, significant differences (p < 0.05) were found in the median volume of the gastric pouch (21 vs. 48 cm(3)), in the percentage of fundus included in the pouch (8.4 vs. 29.4 %), in the complete dissection at the angle of His (10 vs. 3), and in safety parameters. In task 3, significant differences were found in the size and position of enterotomies., Conclusions: The Lap Mentor may be proposed as a certification tool for bariatric surgeons because it also recognizes their specific skills in the technical details of the procedure that affect long-term results. Furthermore, the possibility of analyzing the performance in detail can help define areas where the surgeon is lacking. These findings indicate a potential role of the Lap Mentor in tailoring the training to maximize improvement.

Purpose: The right hemicolectomy may be conducted through laparoscopic or laparotomic surgery, transverse or midline incisions. The transverse laparotomy offers some advantages compared to the midline laparotomy and laparoscopy. A literature review was performed to evaluate the possible advantages of the transverse incision versus midline incision or laparoscopic right hemicolectomy., Methods: A systematic research was performed in Medline, Embase, Cochrane Central Register of Controlled Trials, CINAHL, BioMed Central, and the Science Citation Index., Results: Laparotomic right hemicolectomy with transverse incision is preferable to laparotomic hemicolectomy with midline incision. A transverse incision offers a lessened postoperative pain following physical activity, a lessened need to administer analgesic therapy during the post-operative time, better aesthetic results, and a better post-operative pulmonary function. Open surgery with transverse or midline incision ensured a shorter operative time, lower costs and a greater length of the incision compared to the laparoscopic. However, there are no differences in the oncological outcomes., Conclusions: It was not possible to identify significant differences between the open right hemicolectomy with transverse incision versus the open right hemicolectomy with midline incision or laparoscopic hemicolectomy.

A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.

Fibroblast growth factor receptor-2 (FGFR-2) plays an important role in tumorigenesis. In thyroid cancer it has been observed a FGFR-2 down-modulation, but the role of this receptor has not been yet clarified. Therefore, we decided to examine the expression of both FGFR-2 isoform, FGFR-2-IIIb and FGFR-2-IIIc, in different histological thyroid variants such as hyperplasia, follicular adenoma and papillary carcinoma. Immunohistochemistry and quantitative Real-Time PCR analyses were performed on samples of hyperplasia, follicular adenoma and papillary carcinoma, compared with normal thyroid tissue. Thyroid hyperplasia did not show statistically significant reduction in FGFR-2 protein and mRNA levels. Interestingly, in both follicular adenoma and papillary carcinoma samples we observed a strongly reduced expression of both FGFR-2 isoforms. We speculate that FGFR-2 down-modulation might be an early event in thyroid carcinogenesis. Furthermore, we suggest the potential use of FGFR-2 as an early marker for thyroid cancer diagnosis.

Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers., (© 2013 by the Wound Healing Society.)

Metastatic carcinomas to the thyroid are rare in daily clinical practice. However, when encountered they represent a diagnostic challenge, since it is difficult to distinguish them from primary thyroid lesions, especially when occurring in patients with occult malignant history. Nevertheless, it is critical to differentiate a metastatic tumor from primary thyroid lesions, as the clinical management and the prognosis are different for the two entities. More recently, elastosonography opened new possibilities to ultrasound in different fields, such as thyroid nodule differentiation. Herein, we report a case of metastatic signet ring cell carcinoma to the thyroid studied with quantitative elastography and acoustic radiation force impulse imaging.

Background: Video-games have become an integral part of the new multimedia culture. Several studies assessed video-gaming enhancement of spatial attention and eye-hand coordination. Considering the technical difficulty of laparoscopic procedures, legal issues and time limitations, the validation of appropriate training even outside of the operating rooms is ongoing. We investigated the influence of a four-week structured Nintendo® Wii™ training on laparoscopic skills by analyzing performance metrics with a validated simulator (Lap Mentor™, Simbionix™)., Methodology/principal Findings: We performed a prospective randomized study on 42 post-graduate I-II year residents in General, Vascular and Endoscopic Surgery. All participants were tested on a validated laparoscopic simulator and then randomized to group 1 (Controls, no training with the Nintendo® Wii™), and group 2 (training with the Nintendo® Wii™) with 21 subjects in each group, according to a computer-generated list. After four weeks, all residents underwent a testing session on the laparoscopic simulator of the same tasks as in the first session. All 42 subjects in both groups improved significantly from session 1 to session 2. Compared to controls, the Wii group showed a significant improvement in performance (p<0.05) for 13 of the 16 considered performance metrics., Conclusions/significance: The Nintendo® Wii™ might be helpful, inexpensive and entertaining part of the training of young laparoscopists, in addition to a standard surgical education based on simulators and the operating room.

Retroperitoneal fibrosis (RPF), also known as Ormond's disease, is a rare fibroinflammatory disease with uncertain etiology. RPF is characterized by the presence of a particular retroperitoneal fibrotic tissue which is white, woody and involving retroperitoneal structures such as the great vessels, ureters and psoas muscle. The main complication of RPF is the obstruction of local structures such as the ureters due to the fibrosis and the treatment of this aspect represents the main challenge for this pathology. RPF medical treatment consists of corticosteroids or/and immunosuppressive therapy. We report a case of a patient affected by idiopathic RPF treated with low-dose methotrexate (MTX) and 6-methylprednisolone (6-MP) for two years, describing and confirming the effectiveness and safety of a long-term low-dose MTX and 6-MP treatment associated to ureteral Double-J stenting avoiding more invasive surgical approaches.