FMRP expression in primary breast tumor cells correlates with recurrence and specific site of metastasis.

Breast cancer is the most common cancer among women worldwide. Molecular and clinical evidence indicated that Fragile X Messenger Ribonucleoprotein 1 (FMRP) plays a role in different types of cancer, including breast cancer. FMRP is an RNA binding protein that regulates the metabolism of a large group of mRNAs coding for proteins involved in both neural processes and in epithelial-mesenchymal transition, a pivotal mechanism that in cancer is associated to tumor progression, aggressiveness and chemoresistance. Here, we carried out a retrospective case-control study of 127 patients, to study the expression of FMRP and its correlation with metastasis formation in breast cancer. Consistent with previous findings, we found that FMRP levels are high in tumor tissue. Two categories have been analyzed, tumor with no metastases (referred as control tumors, 84 patients) and tumor with distant metastatic repetition, (referred as cases, 43 patients), with a follow-up of 7 years (mean). We found that FMRP levels were lower in both the nuclei and the cytoplasm in the cases compared to control tumors. Next, within the category cases (tumor with metastases) we evaluated FMRP expression in the specific sites of metastasis revealing a nuclear staining of FMRP. In addition, FMRP expression in both the nuclear and cytoplasmic compartment was significantly lower in patients who developed brain and bone metastases and higher in hepatic and pulmonary sites. While further studies are required to explore the underlying molecular mechanisms of FMRP expression and direct or inverse correlation with the secondary metastatic site, our findings suggest that FMRP levels might be considered a prognostic factor for site-specific metastasis.

PET-guided Switch from Immunotherapy to Targeted Therapy in a Metastatic Melanoma Patient: a personalized approach.

An early identification of non-responders in oncology is of crucial importance to rapidly switch treatment regimens. Here we report a positron emission tomography, (PET)-guided switch from immunotherapy to targeted therapy in a patient affected by metastatic melanoma. We describe the case of a 78-years-old male patient diagnosed with nodular melanoma, submitted to baseline PET/CT with 18fluorodeoxyglucose (18F-FDG) that showed cutaneous and skeletal metastases (stage IV). The patients started immunotherapy with pembrolizumab. A PET/CT performed 3 months after the start of immunotherapy demonstrated progressive metabolic disease both at skeletal and cutaneous level, confirmed also by the biopsy. As patients resulted positive for BRAF V600k mutation, treatment regimen was rapidly switched to combined anti-BRAF/MEK targeted therapy. The PET/CT performed 3 months later, showed almost complete metabolic response. Ten months after the beginning of targeted therapy, the patient continues to present a durable metabolic response. PET/CT with 18F-FDG may help in monitoring the response to treatment in metastatic melanoma thus defining personalized therapeutic pathways.

Autism Spectrum Disorders: Translating human deficits into mouse behavior.

Autism Spectrum Disorders are a heterogeneous group of neurodevelopmental disorders, with rising incidence but little effective therapeutic intervention available. Currently two main clinical features are described to diagnose ASDs: impaired social interaction and communication, and repetitive behaviors. Much work has focused on understanding underlying causes of ASD by generating animal models of the disease, in the hope of discovering signaling pathways and cellular targets for drug intervention. Here we review how ASD behavioral phenotypes can be modeled in the mouse, the most common animal model currently in use in this field, and discuss examples of genetic mouse models of ASD with behavioral features that recapitulate various symptoms of ASD.

A prospective randomized study to compare pelvic floor rehabilitation and dapoxetine for treatment of lifelong premature ejaculation.

Premature ejaculation (PE) is the most common male sexual disorder. We compared pelvic floor muscle rehabilitation to on-demand treatment with the selective serotonin reuptake inhibitor dapoxetine in 40 men with lifelong PE (baseline intra-vaginal ejaculatory latency time (IELT) ≤1 min). Subjects were randomized into the following two treatment groups: (1) PFM rehabilitation or (2) 30 or 60 mg of on-demand dapoxetine. Total treatment time for both groups was 12 weeks, at the end of which, IELT mean values were calculated to compare the effectiveness of the two different therapeutic approaches. At the end of treatment, 11 of the 19 patients (57%) treated with rehabilitation were able to control the ejaculation reflex, with a mean IELT of 126.6 sec (range: 123.6-152.4 sec). In the dapoxetine group, after 12 weeks of therapy, 5 of 8 (62.5%) patients in the 30 mg subgroup and five of seven (72%) in the 60 mg subgroup had an IELT >180 sec (mean: 178.2 and 202.8 sec, respectively). The results obtained in the group treated with pelvic floor rehabilitation are promising, and this treatment represents an important cost reduction if compared to dapoxetine on-demand treatment. The present study confirms the data that are previously available in the literature on the efficacy and safety of the new inhibitor of serotonin reuptake, dapoxetine, as well as proposes and evaluates a new type of physical treatment that may be a viable therapeutic option for treatment of PE.

Expression of phosphoinositide-specific phospholipase C isoforms in human umbilical vein endothelial cells.

AIMS: The signalling system of phosphoinositides (PIs) is involved in a number of cell and tissue functions including membrane trafficking, ion channel activity, cell cycle, apoptosis, differentiation and cell and tissue polarity. Recently, a role in cell migration was hypothesised for PI and related molecules including the phosphoinositide-specific phospholipases C (PI-PLCs), main players in PI signalling. The expression of PI-PLCs is tissue-specific and evidence suggests that it varies under different conditions such as tumour progression or cell activation. In order to obtain a complete picture, the expression of all PI-PLC isoforms was analysed in human endothelial cells (EC). METHODS: Using molecular biology methods (RT-PCR), the expression of PI-PLC isoforms was analysed in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for human EC. RESULTS: All the PI-PLC isoforms except PI-PLC ß1, PI-PLC ε and PI-PLC ζ were expressed in HUVEC. CONCLUSIONS: The growing interest in the complex cascade of events occurring in angiogenesis will provide useful insights for therapeutic strategies. The expression of PI-PLC isoforms in HUVEC is a useful tool for further studies directed to understanding their role in angiogenesis. However, although HUVEC represent a widely used experimental model for human macrovascular EC, limitations remain in that they cannot fully represent the metabolic properties and interactions of the EC distributed in the entire organism.

Purification and characterization of an antifungal thaumatin-like protein from Cassia didymobotrya cell culture.

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

Basal turn cochleostomy via the middle fossa route for cochlear implant insertion.

OBJECTIVE: The current article describes the surgical technique and the very preliminary results of insertion of a cochlear implant, via the middle fossa (MF), in patients with middle ear disease. STUDY DESIGN: The study design was a case report and a description of surgical technique. SETTING: The study was conducted at an ENT Department, University of Verona, Verona, Italy. PATIENTS: Two subjects with profound bilateral hearing loss, the first one presenting a bilateral radical mastoidectomy cavity and the second one with fibroadhesive otitis media, were operated on via the current technique. INTERVENTION: After adequate exposure of the MF floor, a triangular bony area between the greater superficial petrous nerve and the projection of the labyrinthine portion of the facial nerve was drilled out. The basal cochlear turn facing the middle cranial fossa floor was easily encountered, a small cochleostomy measuring 1 1/2 mm in diameter was performed on the most superficial part of the basal turn, and the electrode carrier was inserted into the fenestrated cochlea. The receiver-stimulator was positioned on a bone well drilled previously in the temporal squama. MAIN OUTCOME MEASURES: The activity of the inserted electrodes was tested by means of telemetry and intraoperative recording of the electrically evoked auditory responses. Speech perception tests, performed 15 and 30 days after cochlear implant activation, showed a remarkable improvement in the outcomes versus the preoperative values that are provided for comparison. CONCLUSIONS: This new surgical approach to cochlear implant insertion via the MF route allows stimulation of part of the basal and the middle and apical areas of the cochlea, where greater survival rates of spiral ganglion cells are observed. Cochlear implant insertion via the MF approach represents a promising technique for auditory rehabilitation of subjects with a bilateral radical mastoidectomy cavity, patients suffering from middle ear malformations or chronic middle ear disease due to eustachian tube dysfunction, or subjects with doubtful responses to promontory stimulation.

In vitro study of the NS2-3 protease of hepatitis C virus.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.

Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus.

The hepatitis C virus (HCV) NS3 protease cleaves the viral polyprotein at specific sites to release the putative components of the HCV replication machinery. Selective inhibition of this enzyme is predicted to block virus replication, and NS3 is thus considered an attractive candidate for development of anti-HCV therapeutics. To set up a system for analysis of NS3 protease activity in cultured cells, we constructed a family of chimeric Sindbis viruses which carry sequences coding for NS3 and its activator, NS4A, in their genomes. HCV sequences were fused to the gene coding for the Sindbis virus structural polyprotein via an NS3-specific cleavage site, with the expectation that processing of the chimeric polyprotein, nucleocapsid assembly, and generation of viable viral particles would occur only upon NS3-dependent proteolysis. Indeed, the chimeric genomes encoding an active NS3 protease produced infectious viruses in mammalian cells, while those encoding NS3 inactivated by alanine substitution of the catalytic serine did not. However, in infected cells chimeric genomes recombined, splicing out HCV sequences and reverting to pseudo-wild-type Sindbis virus. To force retention of HCV sequences, we modified one of the initial chimeras by introducing a second NS3 cleavage site in the Sindbis virus portion of the recombinant polyprotein, anticipating that revertants not encoding an active NS3 protease would not be viable. The resulting chimera produced infectious viruses which replicated at a lower rate than the parental construct and displayed a marked temperature dependence in the formation of lysis plaques yet stably expressed NS3.

On the role of agonist-evoked Ca2+ mobilization in sustaining the ongoing phosphoinositide hydrolysis. A study on intact SK-N-BE(2) neuroblastoma cells subjected to muscarinic stimulation.

Stimulation of SK-N-BE(2) cells with 1 mM carbachol (Cch) elicited phosphoinositide (PPI) hydrolysis and a rapid elevation of cytosolic Ca2+ concentration ([Ca2+]i) from 115 nM to about 500 nM, followed by a plateau around 200 nM. In myo [3H]inositol-labelled cells, Cch-evoked accumulation of [3H]inositol phosphate (IPs) was not affected when [Ca2+]i was clamped at resting by cell loading with 10 microM BAPTA/AM; under these conditions, maximal 1,4,5-inositol trisphosphate accumulation was not reduced either. When [Ca2+]i was clamped around 700 nM by cell treatment with 600 nM ionomycin, Cch-evoked [3H]IPs accumulation was enhanced by less than 20%, but it was impaired by a 30% and a 55% after [Ca2+]i reduction to about 70 nM and 35-50 nM, by cell loading with 20 microM or 40 microM BAPTA/AM, respectively. These results show that, in SK-N-BE(2) cells, Cch-activated PPI-specific phospholipase C is sensitive to [Ca2+]i but it already operates under suboptimal conditions at resting [Ca2+]i.

Arachidonic acid modulates [14C]stearic acid incorporation into phosphatidylinositol, in human neuroblastoma cells.

In the human neuroblastoma cell line SK-N-BE(2), arachidonic acid (AA), supplied in the medium at micromolar concentrations, markedly enhanced [14C]stearic acid (SA) (but not [14C]palmitic acid or [14C]oleic acid) incorporation into phosphatidylinositol (PtdIns). AA failed to stimulate [14C]SA incorporation into PtdIns precursors, namely phosphatidic acid and cytidinediphosphodiacylglycerol: furthermore, enhanced [14C]SA incorporation, brought about by exogenously administered AA, was not restricted to PtdIns tetraenoic species. When cells were pulsed for 1 h with [14C]SA (either in the presence or absence of AA) and then reincubated in AA- and [14C]SA-free medium, a marked loss of radioactivity from PtdIns was observed, that however was restricted to molecular species other than tetraenoic. These results are discussed in the light of possible mechanisms through which PtdIns achieves the 1-stearoyl-2-arachidonoyl configuration.

Phosphoinositide-derived diacylglycerol conversion to phosphatidic acid is a receptor-dependent and compartmentalized phenomenon in human neuroblastoma.

We report that upon muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells, the extent of phosphoinositide-derived diacylglycerol (DG) conversion to phosphatidic acid (PA), operated by a DG kinase, is dependent on the potency of receptor stimulation and correlates with the reduction of phosphatidylinositol 4,5-bisphosphate mass. Evidence is provided that agonist-evoked Ca2+ mobilisation or protein kinase activation are not key events in triggering receptor-generated DG conversion to PA; furthermore, the phenomenon is compartmentalized, namely it occurs within a topologically restricted area that is poorly accessible to DG artificially generated by cell treatment with bacterial phosphatidylinositol-specific phospholipase C. Possible mechanisms driving regulation of the DG kinase operating in the transduction system investigated are discussed.

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.

Effects of perphenazine on the metabolism of inositol phospholipids in SK-N-BE(2) human neuroblastoma cells.

Administration of myo-[3H]inositol to SK-N-BE(2) human neuroblastoma cells for 24 hr resulted in equilibrium labelling of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2), as well as in retention of a large intracellular pool of free myo-[3H]inositol. Equilibrium labelling was no longer observed when cells were treated for 2 hr with 20 microM perphenazine (PPZ) in label-free medium; under these conditions, myo-[3H]inositol from the retained intracellular pool was incorporated into PI and PIP but not into PIP2. Analysis of water-soluble myo-[3H]inositol derivatives and inositol 1,4,5-trisphosphate mass determination indicated that PPZ did not stimulate phosphoinositide hydrolysis by phospholipase C. These results indicate that PPZ raises PI and PIP levels, whereas it is ineffective in expanding the PIP2 pool. The latter effect is not due to a concomitant synthesis and hydrolysis of this lipid.

Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation.

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.

Phenothiazines inhibit acetylcholinesterase by concentration-dependent-type kinetics. A study with trifluoperazine and perphenazine.

The properties of perphenazine (PPZ) and trifluoperazine (TFP) as fluorescent dyes were exploited to calculate their critical micellar concentrations. The relative fluorescence quantum yield of the two amphiphiles was dependent on their concentration, abruptly decreasing above 30-40 microM PPZ and 20-30 microM TFP. Evidence is presented that this phenomenon is driven by the formation of non-fluorescent drug aggregates. The type of inhibition kinetics displayed by PPZ and TFP on human erythrocyte acetylcholinesterase (AChE) was also dependent on drug concentration, turning from non-competitive to a "mixed" inhibition type at concentrations at which PPZ and TFP were demonstrated to undergo micelle formation. Results support the notion that phenothiazines may interact with AChE both as monomers and micellar aggregates, producing different inhibitory effects.

Rubidium shows effects different from lithium on phosphatidylinositol metabolism in a cell line of human neuroblastoma.

In a SK-N-BE human neuroblastoma cell line the incubation of rubidium (1 and 10 mM) for 24 h significantly increased IP2 formation, whereas it apparently did not affect other inositol phosphates. In comparison to lithium (10 mM), which significantly enhanced inositolmonophosphate and IP2 accumulation following carbamoylcholine (1 mM) stimulation, rubidium at the same concentration, was unable to affect inositol phosphate accumulation. In conclusion, the present experiments show that rubidium, compared with lithium, shows a different profile on phosphoinositide metabolism since its main action is an increase in phosphatidylinositol turnover. These results may have some relevance to the use of rubidium as antidepressant in man.