Group IV Phospholipase A2α Controls the Formation of Inter-Cisternal Continuities Involved in Intra-Golgi Transport

The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex

Reproducible surface-enhanced Raman quantification of biomarkers in multicomponent mixtures

A.C.D.L. is supported by an AIRC Start-up Grant 11454 and a FIR project RBFR12WAPY. A.D.F. is supported by an EPSRC Career Acceleration Fellowship (EP/I004602/1). D.C. is supported by an AIRC Grant IG10341 and the projects PON01-00117 and PON01-00862. S.M. is supported by a PRIN project 2012CK5RPF_05.Direct and quantitative detection of unlabeled glycerophosphoinositol (GroPIns), an abundant cytosolic phosphoinositide derivative, would allow rapid evaluation of several malignant cell transformations. Here we report label-free analysis of GroPIns via surface-enhanced Raman spectroscopy (SERS) with a sensitivity of 200 nM, well below its apparent concentration in cells. Crucially, our SERS substrates, based on lithographically defined gold nanofeatures, can be used to predict accurately the GroPIns concentration even in multicomponent mixtures, avoiding the preliminary separation of individual compounds. Our results represent a critical step toward the creation of SERS-based biosensor for rapid, label-free, and reproducible detection of specific molecules, overcoming limits of current experimental methods.PostprintPeer reviewe

Faciogenital dysplasia protein Fgd1 regulates invadopodia biogenesis and extracellular matrix degradation and is up-regulated in prostate and breast cancer.

Invadopodia are proteolytically active membrane protrusions that extend from the ventral surface of invasive tumoral cells grown on an extracellular matrix (ECM). The core machinery controlling invadopodia biogenesis is regulated by the Rho GTPase Cdc42. To understand the upstream events regulating invadopodia biogenesis, we investigated the role of Fgd1, a Cdc42-specific guanine nucleotide exchange factor. Loss of Fgd1 causes the rare inherited human developmental disease faciogenital dysplasia. Here, we show that Fgd1 is required for invadopodia biogenesis and ECM degradation in an invasive cell model and functions by modulation of Cdc42 activation. We also find that Fgd1 is expressed in human prostate and breast cancer as opposed to normal tissue and that expression levels matched tumor aggressiveness. Our findings suggest a central role for Fgd1 in the focal degradation of the ECM in vitro and, for the first time, show a connection between Fgd1 and cancer progression, proposing that it might function during tumorigenesis

Biomolecular sensing for cancer diagnostics using highly reproducible SERS substrates

We developed a SERS biosensor based on gold fishnets fabricated by using e-beam lithography. This device is used for glycerophosphoinositol (GroPIns) molecule sensing. GroPIns is an abundant component of cell cytosol and high GroPIns levels have been reported in several tumour cells. We demonstrate that our SERS sensor is able to accurately and quantitatively determine the concentration of GroPIns. These results indicate that SERS may provide a novel platform technology to identify GroPIns profiles in disease pathogenesis.</p

Immunocytochemical detection of hMSH2 and hMLH1 expression in oral SCC.

BACKGROUND: The loss of DNA mismatch repair system was reported in hereditary non-poliposis colon cancer and in other tumours. The aim of this study was to detect the protein expression pattern of hMSH2 and hMLH1 in oral squamous cell carcinoma (SCC) by immunohistochemistry in paraffin-embedded tissues. MATERIALS AND METHODS: 5 specimens, obtained from healthy oral mucosa, and 20 from oral SCC were tested with anti-hMSH2 and anti-hMLH1 monoclonal antibodies. RESULTS: Six cases (30%) showed nuclear positivity in differentiated areas (G1) and cytoplasmic positivity in areas with a lower degree of differentiation, four cases (20%) showed only cytoplasmic positivity, and only one (5%) no staining. One case of oral SCC (5%) showed no hMLH1 staining in the tumoral cells, even if normal squamous epithelium available in this section showed a nuclear positivity; six cases (30%) showed nuclear positivity in differentiated areas (G1) and cytoplasmic positivity in areas with a lower degree of differentiation, three cases (15%) showed only cytoplasmic positivity. CONCLUSIONS: These data suggest that examination of hMSH2 and hMLH1 protein expression by immunohistochemistry is important in oral SCC. The analysis of mismatches expression in these cases of oral SCC might suggest that an absent nuclear staining for both hMSH2 and hML1 could constitute a hallmark of potential phenotype mutator for this type of neoplasia

Immunocytochemical detection of hMSH2 and hMLH1 expression in oral SCC.

BACKGROUND:The loss of DNA mismatch repair system was reported in hereditary non-poliposis colon cancer and in other tumours. The aim of this study was to detect the protein expression pattern of hMSH2 and hMLH1 in oral squamous cell carcinoma (SCC) by immunohistochemistry in paraffin-embedded tissues. MATERIALS AND METHODS:5 specimens, obtained from healthy oral mucosa, and 20 from oral SCC were tested with anti-hMSH2 and anti-hMLH1 monoclonal antibodies. RESULTS:Six cases (30%) showed nuclear positivity in differentiated areas (G1) and cytoplasmic positivity in areas with a lower degree of differentiation, four cases (20%) showed only cytoplasmic positivity, and only one (5%) no staining. One case of oral SCC (5%) showed no hMLH1 staining in the tumoral cells, even if normal squamous epithelium available in this section showed a nuclear positivity; six cases (30%) showed nuclear positivity in differentiated areas (G1) and cytoplasmic positivity in areas with a lower degree of differentiation, three cases (15%) showed only cytoplasmic positivity. CONCLUSIONS:These data suggest that examination of hMSH2 and hMLH1 protein expression by immunohistochemistry is important in oral SCC. The analysis of mismatches expression in these cases of oral SCC might suggest that an absent nuclear staining for both hMSH2 and hML1 could constitute a hallmark of potential phenotype mutator for this type of neoplasia

Paroxysmal nocturnal hemoglobinuria: A long-term single center experience

Introduction: paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of haematopoietic cells characterized by a defect in the glycosylphosphatidylinositol (GPI) anchored molecules. It is characterized by the triad of hemolytic anemia, cytopenias and a high risk of venous thrombosis. In the last 10 years Eculizumab has been employed in the treatment of PNH. Considering the rarity of the disease, so far, related data are the result of multi-center studies. Herein we report the analysis of the largest long-term unicentric series of PNH patients. Patients and Methods: We performed a retrospective analysis in 42 patients (26 female/16 male) followed at our center between February 1985 and September 2016. Median age was 53 years (range 16-79.2). Since 1985 up to 2000 the diagnosis was made by HAM test; starting from 2000, patients were diagnosed by flow cytometry (FC) and the proportion of GPI-AP–deficient granulocytes by FC (clone size) was collected. Moreover, all patients previously diagnosed by Ham test we studied by CF, to confirm the diagnosis and evaluate clone size. During the decade 85-95, 11 patients were diagnosed; 12 during 96-2006; 19 in the last decade. At diagnosis 26 patients had classic PNH, 9 aplastic PNH and 7 intermediate form.1. Results: 30 yrs overall survival (OS) was 84%, censoring, at last follow up, 2 patients dead for non PNH related reasons. In univariate analysis absence of thrombotic events (96% vs 80%) and diagnosis performed during the last decade (100% vs 90% vs 75%) represents factors associated with a better OS, even if statistical significance was not reached. Cumulative incidence of thrombosis, pancytopenia or bicitopenia, and clonal neoplasm were 39%, 18% and 10%, respectively. Up to 2005 treatment options were mainly supportive including blood transfusion or allogenic bone marrow transplantation. Eculizumab was first introduced in 2005 in 4 patients included in Phase III trials (TRIUMPH and SHEPHERD)2, Than the drug has been employed in other 18 patients. Half of patients became transfusion independent, but with Hb level ranging between 8,5 and 11 gr/dl; complete remission was observed in 32%, 18% remained transfusions dependent. A reduction of LDH observed in all patients with improvement of asthenia; no thrombotic event was observed after eculizumab, even if 9 out of 22 patients had recurrent thromboembolisms before the drug. No severe infection were diagnosed, nor renal failure or pulmonary hypertention. One patient developed extravascular hemolysis and receive a succesfully selective splenic artery embolization (SSAE)3 .Ten years OS in Eculizuman group was 92%. No death was due to PNH reason. Discussion: Our unicentric study confirms thrombosis as major complication in PNH patients, and as an important factor influencing OS. We can speculate that the better OS in last decade is due to the use of Eculizumab that reduced thrombotic events. In particular, for patients on eculizumab the curve of OS was 92% at 10 years, even if patients with previous thromboembolism and diagnosis performed before the last decade have been included in this group. Although kidney failure and lung hypertension have been reported in some experiences, we did not observed these complications on a long follow up. We can assume that the availability of a dedicated emergency room allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuric crisis, reducing the risk or organ damage. No infection have been described in our patients. Considering the risk of meningococcus infection we perform a double vaccination with conjugated anti-meningococcus (serotypes ACWY) at least 2 weeks prior to administering the first dose of eculizumab (according by drug schedule) and 2 months after. Moreover, we add Meningococcus B vaccination 3-4 weeks after first dose of drug. However a guideline for vaccination schedule needs to be defined by scientific society

The natural phosphoinositide derivative glycerophosphoinositol inhibits the lipopolysaccharide-induced inflammatory and thrombotic responses

Inflammatory responses are elicited through lipid products of phospholipase A(2) activity that acts on the membrane phospholipids, including the phosphoinositides, to form the proinflammatory arachidonic acid and, in parallel, the glycerophosphoinositols. Here, we investigate the role of the glycerophosphoinositol in the inflammatory response. We show that it is part of a negative feedback loop that limits proinflammatory and prothrombotic responses in human monocytes stimulated with lipopolysaccharide. This inhibition is exerted both on the signaling cascade initiated by the lipopolysaccharide with the glycerophosphoinositol-dependent decrease in IB kinase /, p38, JNK, and Erk1/2 kinase phosphorylation and at the nuclear level with decreased NF-B translocation and binding to inflammatory gene promoters. In a model of endotoxemia in the mouse, treatment with glycerophosphoinositol reduced TNF- synthesis, which supports the concept that glycerophosphoinositol inhibits the de novo synthesis of proinflammatory and prothrombotic compounds and might thus have a role as an endogenous mediator in the resolution of inflammation. As indicated, this effect of glycerophosphoinositol can also be exploited in the treatment of manifestations of severe inflammation by exogenous administration of the compound