PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Detection of an asymptomatic right-ventricle cardiac metastasis from a small-cell lung cancer by F-18-FDG PET/CT.

A right heart metastasis of a small-cell lung cancer was found on the whole-body F-fluoro-deoxy-glucose positron emission tomography/computed tomography (F-FDG-PET/CT) of a 69-year-old smoker investigated for a right pulmonary mass discovered on chest radiography after a fracture of the right humerus. The PET scan showed an increased FDG uptake by the mass in the right lung and an intense, atypical focal activity of the right ventricle strongly suggestive of a neoplastic process. CT-guided lung biopsy revealed a small-cell lung cancer and myocardial biopsy confirmed the presence of a cardiac metastasis. The patient was treated with six cycles of chemotherapy followed by radiation therapy, which included the heart lesion. At follow-up PET/CT 2 months after the end of treatment, the abnormal cardiac uptake had disappeared, whereas increased FDG uptake persisted in the pulmonary residual mass.

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells.

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

Owl pellets as a source of DNA for genetic studies of small mammals.

Owl pellets contain a good skeletal record of the small mammals consumed, and correspond to the undigested portions of prey which are regurgitated. These pellets are easy to find at the roosting site of owls. As it has been demonstrated that amplifiable DNA can be isolated from ancient bone remains, the possibility of using owl pellets as a source of DNA for small mammal genetics studies via the polymerase chain reaction has been investigated. The main uncertainties when isolating DNA from such a material are firstly the possibility that the extracted DNA would be too degraded during the digestion in the stomach of the owl, and secondly that extensive cross-contaminations could occur among the different prey consumed. The results obtained clearly demonstrate that cross-contamination does not occur, and that mitochondrial and nuclear DNA can be amplified using skulls of small mammals found in owl pellets as a source of DNA. The relative efficiency of two methods of DNA extraction is estimated and discussed. Thus, owl pellets represent a non-invasive sampling technique which provides a valuable source of DNA for studying population genetics of small mammals.

Proteasomal cleavage does not determine immunogenicity of gp100-derived peptides gp100 209-217 and gp100 209-217T210M.

Immune responses against tumor-associated antigens rely on efficient epitope presentation. The melanoma-associated antigen (Ag) gp100 contains HLA-A*0201 ligands that are characterized by low to medium binding affinity, among which gp100(209-217) is the most prominent (Kawakami et al., J Immunol 154:3961-3968, 1995). While this epitope is a natural T-cell target, it primes with low-efficiency T-cell responses during immunization. A modified gp100 epitope, gp100(209-217T210M), that contains a Thr to Met substitution at position 2 of the antigenic nonamer is characterized by high binding affinity for HLA-A*0201 and elicits strong and clinically effective T-cell responses. This higher affinity is believed to represent the sole reason for enhanced immunogenicity. Contrasting with this observation is the unpredictable relationship between affinity and immunogenicity observed in other antigen systems. In addition, we noted a striking difference between the capability of endogenously processed gp100(209-217) and gp100(209-217T210M) to induce T-cell responses in an in vitro model. Therefore, we questioned whether factors other than HLA-affinity might play a role in determining the immunogenicity of these epitopes. In the present study, we evaluated the in vitro proteasomal cleavages of 23meric precursor peptides encompassing the native sequence (gp100(201-223)) or the modified sequence (gp100(201-223T210M)). Here we show that the standard proteasome liberates the C-termini of both antigenic peptides but not the N-termini. Quantitative analysis of the digestion products revealed that more of the fragments displaying the final C-termini were produced from the wild-type precursor. However, a stronger TCR engagement was observed when fractions of digested gp100(201-223T210M) were used to activate an HLA-A*0201-expressing target T-cell clone. This difference was also found using separately produced, synthetic nonamers. In conclusion, the high binding affinity of gp100(209-217T210M) seems to compensate for possible differences in proteasomal cleavage at the biological level. Since the final antigenic nonamer is not directly produced by the proteasome, additional further factors may influence the antigenic peptide availability, such as post-proteasomal processing and intracellular peptide transport.

Effect of a small dose of alcohol on the endurance performance of trained cyclists.

AIM: The aim of this study was to investigate the effect of an acute small ethanol (EtOH) dose (0.5 ml EtOH/kg fat-free mass, combined with carbohydrate) in a drink on endurance performance of trained cyclists. METHODS: Thirteen well-trained male cyclists took part in this study. A 60-min cycling endurance performance test (time trial) was performed in a calorimetric chamber after drinking an EtOH (30 +/- 1.8 ml) or a non-EtOH control (C) drink. RESULTS: Overall, EtOH induced a significant decrease in the average cycling power output (PO) (EtOH: 233 +/- 23 W versus C: 243 +/- 24 W, P < 0.01). The time course of mechanical PO showed an early decrease during the EtOH trial as compared to C (P < 0.01). Due to the lower PO, oxygen consumption, carbon dioxide production and glucose oxidation were significantly lower (P < 0.05) as compared to C. Relative to PO, heart rate response and ratings of perceived exertion (RPE) were increased by EtOH as compared to C (P < 0.05). In contrast, EtOH did not influence gross work efficiency, glycaemia and blood lactate concentration. CONCLUSIONS: These results show that the acute low dose of EtOH decreased endurance performance. An increase of cardio-vascular strain and psychobiological mechanisms may explain this decrease of endurance performance.

Virosome-Formulated Plasmodium falciparum AMA-1 & CSP Derived Peptides as Malaria Vaccine: Randomized Phase 1b Trial in Semi-Immune Adults & Children.

Background: This trial was conducted to evaluate the safety and immunogenicity of two virosome formulated malaria peptidomimetics derived from Plasmodium falciparum AMA-1 and CSP in malaria semi-immune adults and children.Methods: The design was a prospective randomized, double-blind, controlled, age-deescalating study with two immunizations. 10 adults and 40 children (aged 5-9 years) living in a malaria endemic area were immunized with PEV3B or virosomal influenza vaccine Inflexal (R) V on day 0 and 90.Results: No serious or severe adverse events (AEs) related to the vaccines were observed. The only local solicited AE reported was pain at injection site, which affected more children in the Inflexal (R) V group compared to the PEV3B group (p = 0.014). In the PEV3B group, IgG ELISA endpoint titers specific for the AMA-1 and CSP peptide antigens were significantly higher for most time points compared to the Inflexal (R) V control group. Across all time points after first immunization the average ratio of endpoint titers to baseline values in PEV3B subjects ranged from 4 to 15 in adults and from 4 to 66 in children. As an exploratory outcome, we found that the incidence rate of clinical malaria episodes in children vaccinees was half the rate of the control children between study days 30 and 365 (0.0035 episodes per day at risk for PEV3B vs. 0.0069 for Inflexal (R) V; RR = 0.50 [95%-CI: 0.29-0.88], p = 0.02).Conclusion: These findings provide a strong basis for the further development of multivalent virosomal malaria peptide vaccines.

Small GTPase RhoE/Rnd3 is a critical regulator of Notch1 signaling.

Aberrations of Notch signaling have been implicated in a variety of human cancers. Oncogenic mutations in NOTCH1 are common in human T-cell leukemia and lymphomas. However, loss-of-function somatic mutations in NOTCH1 arising in solid tumors imply a tumor suppressor function, which highlights the need to understand Notch signaling more completely. Here, we describe the small GTPase RhoE/Rnd3 as a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) that arise in skin epithelia. RhoE is a transcriptional target of activated Notch1, which is attenuated broadly in SCC cells. RhoE depletion suppresses Notch1-mediated signaling in vitro, rendering primary keratinocytes resistant to Notch1-mediated differentiation and thereby favoring a proliferative cell fate. Mechanistic investigations indicated that RhoE controls a key step in Notch1 signaling by mediating nuclear translocation of the activated portion of Notch1 (N1IC) through interaction with importins. Our results define RhoE as a Notch1 target that is essential for recruitment of N1IC to the promoters of Notch1 target genes, establishing a regulatory feedback loop in Notch1 signaling. This molecular circuitry may inform distinct cell fate decisions to Notch1 in epithelial tissues, where carcinomas such as SCC arise. Cancer Res; 74(7); 2082-93. ©2014 AACR.

Synthesis of a non-peptidic PET tracer designed for α5β1 integrin receptor.

Arginine-glycine-aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α5 β1 integrin receptor have been developed with promising results. Sixty-one antagonists were screened, and tert-butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α5 β1 integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide-alkyne copper(II)-catalyzed Huisgen's cycloaddition by using 1-azido-2-[(18)F]fluoroethane ([(18)F]12). Different reaction conditions between PMt and [(18)F]12 were investigated, but all of them afforded [(18)F]FPMt in 15 min with similar radiochemical yields (80-83%, decay corrected). Overall, the final radiopharmaceutical ([(18)F]FPMt) was obtained after a synthesis time of 60-70 min in 42-44% decay-corrected radiochemical yield.

A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317-326 Peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

A realistic Computed Tomography simulator for small motion analysis of cerebral aneurysms.

This paper describes a realistic simulator for the Computed Tomography (CT) scan process for motion analysis. In fact, we are currently developing a new framework to find small motion from the CT scan. In order to prove the fidelity of this framework, or potentially any other algorithm, we present in this paper a simulator to simulate the whole CT acquisition process with a priori known parameters. In other words, it is a digital phantom for the motion analysis that can be used to compare the results of any related algorithm with the ground-truth realistic analytical model. Such a simulator can be used by the community to test different algorithms in the biomedical imaging domain. The most important features of this simulator are its different considerations to simulate the best the real acquisition process and its generality.