Identification Of Novel Mechanisms Of Glucolipotoxicity In Type 2 Diabetes.

PhDType 2 Diabetes, a metabolic disorder associated with chronic hyperglycaemia and hyperlipidaemia, is characterised by an impairment of insulin secretion and production and β-cell death. This β-cell dysfunction is determined by different factors, among which inflammatory processes, characterised by increased expression of pro-inflammatory cytokines and chemokines. Although some molecular mechanisms have been proposed to be involved in this β-cell dysfunction, they fail to explain the whole process. In this thesis, a combined approach of microarray, RNAseq, RT-qPCR and western blot will be used to elucidate the pathways affected under glucolipotoxicity, in order to discover novel molecules involved in the pathogenesis of T2D. We found that INS-1 cells exposed to 27 mM glucose, 200 μM oleic acid, 200 μM palmitic acid, show an overexpression of CD40, a TNF receptor involved in inflammation (more than 300% p<0.01), both at RNA and protein level. These data were validated in cultured human islets (p<0.05) and in islets of mice fed a high fat diet (p<0.05). We showed also that siRNA downregulation of CD40 is associated with increase in insulin secretion (p<0.05), revealing a potential new role of this receptor in β-cells. In addition, RNAseq analysis revealed a wide list of molecules differentially expressed in glucolipotoxicity, in particular molecules involved in inflammation, insulin/IGF pathway, fatty acids-cholesterol metabolism and biosynthesis. We focused our attention on potential novel targets, including the thyroid pathway, unknown microRNAs and novel genes, in order to discover new pathways involved in the impairment of insulin secretion in T2D. This work will open the way to future studies aiming to characterise these molecules and to understand their role in the insulin secretion process. Interesting candidates can then be used in the future as potential targets for the development of new and specific therapeutic strategies

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease

: Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors

Clinical Mass Spectrometry in Immunosuppressant Analysis: Toward a Full Automation?

The analysis of immunosuppressive drugs allows the physician to monitor, and eventually correct, immunosuppressive therapy. The panel of molecules under evaluation includes cyclosporine A (CsA), tacrolimus, sirolimus, and everolimus. Initially, assays were performed by immunometric methods, but in the past few years this methodology has been largely superseded by a more accurate and specific technique, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which is now considered the &ldquo;gold standard&rdquo; for immunosuppressant analysis. Both LC-MS/MS and often also immunoassays require a preanalytical manual sample preparation, which involves time-consuming sequential operations whose traceability is often hampered and adds up to the probability of gross errors. The aim of this work was to compare an &ldquo;open&rdquo; LC-MS/MS with a fully automated system, consisting of LC instrumentation combined with a triple quadrupole MS, named Thermo ScientificTM CascadionTM SM Clinical Analyzer (Cascadion). Such automated systems suit the requirements of the reference method and are designed to completely eliminate all of the manual procedures. More than 2000 immunosuppressant samples were analyzed both with the open LC-MS/MS and with Cascadion. Statistics allowed the evaluation of linearity, intra- and inter-assay CV%, bias %, limit of detection and of quantitation, and Passing&ndash;Bablok and Bland&ndash;Altman plots. Results indicated a good correlation between the two methods. In both cases, methods confirmed their suitability for diagnostic settings. Cascadion could provide support when the presence of specialized personnel is lacking, and/or when great productivity and continuous workflow are required

Optimization of the bio-functionalized area of magnetic biosensors

In this work, a new method to functionalize a gold surface by dip coating with a functional copolymer is presented. The coating procedure is simple, robust and can be accomplished in less than one hour. Atomic force microscopy (AFM) scratch tests reveal the presence of a homogeneous polymer coating with a thickness of 2.5nm. X-ray photoemission spectroscopy spectra from C1s, N1s and O1s levels present the typical fingerprints of the polymeric overlayer, i.e. the characteristic peaks from CNC   O and NC   O groups. Surface plasmon resonance (SPR) binding assays were used to check the coating functional properties. Immobilization of heparin to SPR gold surfaces functionalized with copoly(DMA-NAS-MAPS)- followed by binding analysis with the well known heparin binding protein fibroblast growth factor 2 yield binding kinetic parameters comparable to those obtained with commercially available carboxymethyl dextran- functionalized sensorchips, thus confirming the great potential of the proposed technique

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells

Process analysis of pluripotent stem cell differentiation to megakaryocytes to make platelets applying European GMP.

Quality, traceability and reproducibility are crucial factors in the reliable manufacture of cellular therapeutics, as part of the overall framework of Good Manufacturing Practice (GMP). As more and more cellular therapeutics progress towards the clinic and research protocols are adapted to comply with GMP standards, guidelines for safe and efficient adaptation have become increasingly relevant. In this paper, we describe the process analysis of megakaryocyte manufacture from induced pluripotent stem cells with a view to manufacturing in vitro platelets to European GMP for transfusion. This process analysis has allowed us an overview of the entire manufacturing process, enabling us to pinpoint the cause and severity of critical risks. Risk mitigations were then proposed for each risk, designed to be GMP compliant. These mitigations will be key in advancing this iPS-derived therapy towards the clinic and have broad applicability to other iPS-derived cellular therapeutics, many of which are currently advancing towards GMP-compliance. Taking these factors into account during protocol design could potentially save time and money, expediting the advent of safe, novel therapeutics from stem cells

A cutting-edge approach based on UHPLC-MS to simultaneously investigate oxysterols and cholesterol precursors in biological samples: Validation in Huntington's disease mouse model

Brain is most cholesterol-rich organ in the body. Since cholesterol does not cross the blood brain barrier, its metabolism is provided in situ by astrocytes and neurons, and it is crucial for maintaining sterol levels and neuronal integrity and function. Recent studies have shown that the levels of cholesterol precursors and metabolites are lower in the brains of animal models of Huntington's disease (HD) while reduced levels of its catabolite are detected in the plasma of patients. In this study, we introduce a novel analytical method designed to fulfill the complex analytical requirements associated with cholesterol metabolites detection in neurodegenerative disorders. The method allows for the simultaneous quantification of a specific set of oxysterols along with cholesterol precursors in biological samples.The proposed method uses an Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) system operating in multiple reaction monitoring (MRM). Since sterols can be found in biological matrices in either free form or esterified to various fatty acids, a three-step extraction procedure was devised, consisting of alkaline hydrolysis, liquid-liquid extraction and final concentration omitting the need for a solid-phase extraction (SPE) step.The validated method achieved a detection limit of 10 ng/mL in plasma and 1 ng/mg in brain tissue, reaching a comparable sensitivity to previously published LC-MS and GC–MS methods. All target analytes were separated on a reverse-phase column employing a segmented gradient and a temperature ramp. This strategy enabled the elution and separation of all selected metabolites within a 30-minutes timeframe. This innovative approach was employed to quantify cholesterol metabolites in both plasma and brain samples from wild-type (WT) and R6/2 mice, a mouse model of HD. The results obtained from the sample analysis highlighted a significant reduction in desmosterol levels in the R6/2 brain at 12 weeks.In conclusion, the proposed method paves the way for further development of high-sensitive and reproducible protocols to comprehensively investigate simultaneous alterations in both cholesterol biosynthesis and catabolism in HD samples

Systems genetics identifies a macrophage cholesterol network associated with physiological wound healing.

Among other cells, macrophages regulate the inflammatory and reparative phases during wound healing but genetic determinants and detailed molecular pathways that modulate these processes are not fully elucidated. Here, we took advantage of normal variation in wound healing in 1,378 genetically outbred mice, and carried out macrophage RNA-sequencing profiling of mice with extreme wound healing phenotypes (i.e., slow and fast healers, n = 146 in total). The resulting macrophage coexpression networks were genetically mapped and led to the identification of a unique module under strong trans-acting genetic control by the Runx2 locus. This macrophage-mediated healing network was specifically enriched for cholesterol and fatty acid biosynthetic processes. Pharmacological blockage of fatty acid synthesis with cerulenin resulted in delayed wound healing in vivo, and increased macrophage infiltration in the wounded skin, suggesting the persistence of an unresolved inflammation. We show how naturally occurring sequence variation controls transcriptional networks in macrophages, which in turn regulate specific metabolic pathways that could be targeted in wound healing

Insights into kinetics, release, and behavioral effects of brain-targeted hybrid nanoparticles for cholesterol delivery in Huntington's disease

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment