Adolescent amphetamine exposure elicits dose-specific effects on monoaminergic neurotransmission and behaviour in adulthood.

Despite the growing non-medical consumption of amphetamine (Amph) during adolescence, its long-term neurobiological and behavioural effects have remained largely unexplored. The present research sought to characterize the behavioural profile and electrophysiological properties of midbrain monoaminergic neurons in adult rodents after Amph exposure during adolescence. Adolescent rats were administered vehicle, 0.5, 1.5, or 5.0 mg/kg.d Amph from postnatal day (PND) 30–50. At adulthood (PND 70), rats were tested in an open-field test (OFT) and elevated plus maze (EPM), paralleled by in-vivo extracellular recordings of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) neurons from the dorsal raphe nucleus, ventral tegmental area, and locus coeruleus, respectively. 5-HT firing in adulthood was increased in rats that had received Amph (1.5 mg/kg.d) during adolescence. At this regimen, DA firing activity was increased, but not NE firing. Conversely, the highest Amph dose regimen (5.0 mg/kg.d) enhanced NE firing, but not DA or 5-HT firing rates. In the OFT, Amph (1.5 mg/kg.d) significantly increased the total distance travelled, while the other doses were ineffective. In the EPM, all three Amph doses increased time spent in the open arms and central platform, as well as the number of stretch-attend postures made. Repeated adolescent exposure to Amph differentially augments monoaminergic neuronal firing in a dose-specific fashion in adulthood, with corresponding alterations in locomotion, risk assessment (stretch-attend postures and central platform occupancy) and risk-taking behaviours (open-arm exploration). Thus, adolescent Amph exposure induces long-lasting neurophysiological alterations that may have implications for drug-seeking behaviour in the future

Quantification de la translocation membranaire et de l'échappement endosomal : une approche de biologie chimique

Malgré les grands progrès dans la mise au point d'outils thérapeutiques, il reste encore beaucoup de difficultés à surmonter pour mettre au point des modalités de traitement contre le cancer, les plus efficaces possibles. Au cours de ces dernières années, le processus cellulaire d'échappement endosomal, ou plus généralement de translocation membranaire, est devenu un sujet d'intérêt, et sa faible efficacité apparaît aujourd'hui comme le principal obstacle au développement de nouvelles médicaments macromoléculaires, telles que les conjugués protéiques, les nanoparticules oligonucléotides thérapeutiques etc. En effet, en se liant à la surface cellulaire, la plupart des transporteurs thérapeutiques intériorisés ne traversent pas spontanément les membranes cellulaires, restant piégés dans la voie endocytaire. Jusqu'à présent, malgré un effort remarquable, peu de technologies et de stratégies ont été décrites pour favoriser efficacement la translocation des endosomes du lumen vers le cytosol. L'une des raisons qui ralenti les progrès d’élaboration de stratégies efficaces pour favoriser l'échappement endosomal, réside dans la difficulté de quantifier avec précision l'étape de translocation au niveau de la membrane et donc de disséquer mécaniquement le processus. Par conséquence, la première partie de mon doctorat était focalisée sur le développement d'un test universel, quantitatif, sensible et robuste, visant l'investigation et la quantification du processus d'échappement endosomal. Dans la deuxième partie, j'ai appliqué le nouvel essai pour étudier et quantifier la translocation membranaire de la sous-unité B de la toxine Shiga (STxB). Objectif 1 : Sur la base d'une analyse précise des tests actuellement disponibles et de la compréhension complète de leurs inconvénients, nous avons développé un système robuste et sensible qui permet de quantifier la translocation cytosolique pour un porteur donné. Notre stratégie repose sur la modification d'un porteur donné qui se produit exclusivement dans le cytosol de la cellule et qui permet de discriminer, d'isoler et puis de quantifier, la fraction du porteur qui a été transloquée dans le cytosol. La modification cytosolique est effectuée par un système de marquage qui s'exprime exclusivement dans le cytosol et comprend la fusion de deux protéines. La première, conventionnellement appelée Tag 1, est responsable de la liaison covalente et irréversible du porteur transloqué. La seconde, Tag 2, est exploitée comme crochet pour isoler la fraction marquée, permettant ainsi sa quantification. Les protéines SNAP-tag, HaloTag et NeonGreen ont été choisies comme Tags potentiels, et leur activité a d'abord été évaluée in vitro, à l'aide de modèles simplifiés, puis corroborée dans un système cellulaire, en s'assurant que chaque étape se déroulait avec une cinétique rapide et une production quantitative. La protéine SNAP-tag a été identifiée comme le meilleur candidat pour le Tag 1, tandis que la protéine NeonGreen représente le Tag 2, c’est à dire le crochet pour isoler la fraction cytosolique. Le système de marquage cytosolique SNAP-NeonGreen a permis le développement d'un test très sensible, capable de détecter des quantités infimes de protéines transloquées, jusqu'à la plage picomolaire. La protéine de fusion SNAP-NeonGreen a été mise en œuvre dans une lignée cellulaire cancéreuse monoclonale stable pour l'évaluation et la quantification de l'efficacité de translocation du STxB. Objectif 2 : La première application du nouvel essai a été d'étudier la capacité de translocation du STxB. Nous montrons d'abord que la translocation du STxB dépend du récepteur, du temps et de la concentration, et qu'elle est supprimée dans des conditions de privation d’ATP, ainsi que d'incubation à basse température. Pour la première fois, nous avons ensuite fourni une quantification absolue de la translocation du STxB à 37°C, sur une période de 4 heures, dans le modèle cellulaire HeLa.Objectives of the project despite great progress in the development of therapeutic delivery tools, significant difficulties still need to be overcome for the development of maximally efficient cancer treatment modalities. In the past few years, the cellular process of endosomal escape, or more in general membrane translocation, has become a focus of interest, and its low efficiency now appears as the main obstacle for the development of new macromolecular therapeutics, such as protein conjugates, therapeutic oligonucleotide nanoparticles etc. Indeed, upon binding to the cell surface, most of the internalized therapeutic carriers fail to cross cellular membranes spontaneously, remaining trapped within the endocytic pathway. Up to now, despite the remarkable effort, only few technologies and strategies have been described that can efficiently promote the translocation from the lumen of endosomes to the cytosol. One of the reasons for the slow progress in developing efficient strategies to foster endosomal escape resides in the difficulty to accurately quantify the membrane translocation step and therefore to mechanistically dissect the process. Hence, the first part of my PhD was focused on the development of a universal, quantitative, sensitive, and robust assay aimed at the investigation and quantification of the endosomal escape process. In the second part, I have applied the newly developed assay to study and quantify the membrane translocation of the engineered therapeutic carrier Shiga toxin B-subunit (STxB). Objective 1: Engineering a cytosolic tag Based on an accurate analysis of the currently available assays and the full understanding of their drawbacks, we developed a robust and sensitive system that allows the quantification of the cytosolic translocation for a given carrier. To avoid artefacts and limitations of the previously described assays, we excluded subcellular fractionation procedures and the use of fluorescence ligands, FACS and Western blotting as readout methods. Indeed, our strategy relies on modification of a given carrier that exclusively occurs within the cytosol of the cell and that allows to discriminate, isolate, and subsequently quantify the fraction of the carrier that was translocated to the cytosol. The cytosolic modification is carried out by a tag system that is exclusively expressed in the cytosol and comprises the fusion of two proteins. The first, conventionally referred as Tag 1, is responsible to covalently and irreversibly binding the translocated carrier, while the second, Tag 2, is exploited as hook to isolate the tagged fraction, thereby allowing its quantification. SNAP-tag, HaloTag and NeonGreen protein have been chosen as potential Tags, and their activity has been evaluated first in vitro, using simplified models, and then corroborated in a cellular system, making sure that each step occurred with rapid kinetics and quantitative yield. SNAP-tag protein has been identified as the best candidate for Tag 1, whereas NeonGreen protein represents Tag 2, namely the hook to isolate the cytosolic fraction. The cytosolic SNAP-NeonGreen tag system allowed the development of a highly sensitive assay, able to detect minute amounts of translocated protein, down to the picomolar range. The SNAP-NeonGreen fusion protein has been implemented in a monoclonal stable cancer cell line for the evaluation and quantification of STxB translocation efficiency. Objective 2: Evaluation of STxB translocation The first application of the newly developed assay was to investigate the translocation capacity of STxB. We first show that STxB translocation is receptor, time, and concentration dependent, and is abolished in ATP-depletion and low temperature incubation conditions. For the first time, we then provided an absolute quantification of STxB translocation at 37°C, over a period of 4 hours, in HeLa cell

High-dose vitamin B supplementation for persistent visual deficit in multiple sclerosis: a pilot study

The aim of this study is to investigate the potential neuroprotective effect of high-doses vitamins B1, B6 and B12 in patients with relapsing-remitting multiple sclerosis (RRMS) and persistent visual loss after acute optic neuritis (AON). Sixteen patients (20 eyes) diagnosed with RRMS and visual permanent disability following AON were enrolled for the present open, pilot study. Each patient was treated with oral high-doses 300 mg of vitamin B1, 450 mg of vitamin B6 and 1,500 mcg of vitamin B12, as add-on treatment to concomitant disease-modifying therapies (DMTs) for consecutive 90 days. Outcome measures were to determine changes from baseline to month three in visual acuity (VA) and visual field (VF) testing, with correlations with clinical parameters. Logistical regression was performed to evaluate predictors of final VA. A statistically significant improvement was registered in visual acuity (p = 0.002) and foveal sensitivity threshold (FT) (p = 0.006) at follow-up compared to baseline. A similar trend was demonstrated for mean deviation (MD) (p < 0.0001), and pattern standard deviation (PSD) (p < 0.0001). Age at the time of inclusion was positively correlated with latency time (rho = 0.47, p = 0.03), while showing a negative correlation with visual acuity (rho = - 0.45, p = 0.04) and foveal sensitivity threshold (rho = - 0.6, p = 0.005) at follow up. A statistically significant correlation was demonstrated between foveal sensitivity threshold and visual acuity at baseline (rho = 0.79, p < 0.0001). In a linear regression model, the main predictor of visual acuity at follow up was the foveal sensitivity threshold (B = 1.39; p < 0.0001). Supplemental high-dose vitamins B1, B6 and B12 resulted as effective therapy to improve visual function parameters in MS-related visual persistent disability

Absolute Quantification of Drug Vector Delivery to the Cytosol

International audienceMacromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process

A predictive score for optimal cytoreduction at interval debulking surgery in epithelial ovarian cancer: a two- centers experience

Abstract Background Optimal cytoreduction (macroscopic Residual Tumor, RT = 0) is the best survival predictor factor in epithelial ovarian cancer (EOC). It doesn’t exist a consolidated criteria to predict optimal surgical resection at interval debulking surgery (IDS). The aim of this study is to develop a predictive model of complete cytoreduction at IDS. Methods We, retrospectively, analyzed 93 out of 432 patients, with advanced EOC, underwent neoadjuvant chemotherapy (NACT) and IDS from January 2010 to December 2016 in two referral cancer centers. The correlation between clinical-pathological variables and residual disease at IDS has been investigated with univariate and multivariate analysis. A predictive score of cytoreduction (PSC) has been created by combining all significant variables. The performance of each single variable and PSC has been reported and the correlation of all significant variables with progression free survival (PFS) has been assessed. Results At IDS, 65 patients (69,8%) had complete cytoreduction with no residual disease (R = 0). Three criteria independently predicted R > 0: age ≥ 60 years (p = 0.014), CA-125 before NACT > 550 UI/dl (p = 0.044), and Peritoneal Cancer Index (PCI) > 16 (p  16, a PSC ≥ 3 and the presence of R > 0 after IDS were all significantly associated with shorter PFS (p  0). The PSC should be prospectively validated in a larger series of EOC patients undergoing NACT-IDS

Microfluidics for 3D Cell and Tissue Cultures: Microfabricative and Ethical Aspects Updates

The necessity to improve in vitro cell screening assays is becoming ever more important. Pharmaceutical companies, research laboratories and hospitals require technologies that help to speed up conventional screening and therapeutic procedures to produce more data in a short time in a realistic and reliable manner. The design of new solutions for test biomaterials and active molecules is one of the urgent problems of preclinical screening and the limited correlation between in vitro and in vivo data remains one of the major issues. The establishment of the most suitable in vitro model provides reduction in times, costs and, last but not least, in the number of animal experiments as recommended by the 3Rs (replace, reduce, refine) ethical guiding principles for testing involving animals. Although two-dimensional (2D) traditional cell screening assays are generally cheap and practical to manage, they have strong limitations, as cells, within the transition from the three-dimensional (3D) in vivo to the 2D in vitro growth conditions, do not properly mimic the real morphologies and physiology of their native tissues. In the study of human pathologies, especially, animal experiments provide data closer to what happens in the target organ or apparatus, but they imply slow and costly procedures and they generally do not fully accomplish the 3Rs recommendations, i.e., the amount of laboratory animals and the stress that they undergo must be minimized. Microfluidic devices seem to offer different advantages in relation to the mentioned issues. This review aims to describe the critical issues connected with the conventional cells culture and screening procedures, showing what happens in the in vivo physiological micro and nano environment also from a physical point of view. During the discussion, some microfluidic tools and their components are described to explain how these devices can circumvent the actual limitations described in the introduction

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation

International audienceThe adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology

International audienc

Estimating minimum adult HIV prevalence: A cross-sectional study to assess the characteristics of people living with HIV in Italy

In 2012, we conducted a retrospective cross-sectional study to assess the number of people living with HIV linked to care and, among these, the number of people on antiretroviral therapy. The health authority in each of the 20 Italian Regions provided the list of Public Infectious Diseases Clinics providing antiretroviral therapy and monitoring people with HIV infection. We asked every Public Infectious Diseases Clinic to report the number of HIV-positive people diagnosed and linked to care and the number of those on antiretroviral therapy during 2012. In 2012, 94,146 people diagnosed with HIV and linked to care were reported. The majority were males (70.1%), Italians (84.4%), and aged between 25 and 49 years (63.4%); the probable route of transmission was heterosexual contact in 37.5% of cases, injecting drug use in 28.1%, and male-to-male contact in 27.9%. Among people in care, 20.1% had less than 350 CD4 cells/μl, 87.6% received antiretroviral therapy, and among these, 62.4% had a CD4 cell count higher than 350 cells/μl. The overall estimated prevalence of individuals diagnosed and linked to care in 2012 in Italy was 0.16 per 100 residents (all ages). Adding the estimated proportion of undiagnosed people, the estimated HIV prevalence would range between 0.19 and 0.26 per 100 residents. In Italy, the majority of people diagnosed and linked to care receive antiretroviral therapy. A higher prevalence of individuals diagnosed and linked to care was observed in Northern Italy and among males. More information for developing the HIV care continuum is necessary to improve the entire engagement in care, focusing on test-and-treat strategies to substantially reduce the proportion of people still undiagnosed or with a detectable viral load