Skeletal-Muscle Glutamine Synthase is Upregulated in Preclinical Prion Diseases

In prion diseases, aggregates of misfolded prion protein (PrP$^{Sc}$) accumulate not only in the brain but can also be found in various extraneural tissues. This raises the question whether prion-specific pathologies arise also in these tissues. Here we sequenced mRNA transcripts in skeletal muscle, spleen and blood of prion-inoculated mice at eight timepoints during disease progression. We detected consistent gene-expression changes in all three organs, with skeletal muscle showing the most uniform alterations during disease progression. The glutamate synthetase (GLUL) gene was monotonically upregulated in skeletal muscle of mice infected with three different scrapie prion strains (RML, ME7 and 22L) and in human sporadic Creutzfeldt-Jakob disease. GLUL dysregulation was accompanied by changes in glutamate/glutamine metabolism, leading to reduced glutamate levels in skeletal muscle. None of these changes were observed in skeletal muscle of humans with amyotrophic lateral sclerosis, Alzheimer’s disease, or dementia with Lewy bodies, suggesting that they are specific to prion diseases. Besides pointing to unrecognized metabolic implications of prion infections, these findings suggest that GLUL could represent an accessible biomarker of prion disease progression, particularly during the preclinical stages of disease, and might be useful for monitoring the efficacy of experimental antiprion therapies

Concomitant Administration of Capecitabine and Folate Supplements: Need to Encourage Medication Reconciliation

Hand-Foot syndrome (HFS) and diarrhoea are dose-limiting Adverse Drug Reactions (ADRs) of capecitabine-based chemotherapy. Four polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) gene, encoding the DPD enzyme responsible for the metabolism of fluoropyrimidines, such as capecitabine, are strongly associated with severe ADRs, and their screening should be performed before starting treatment. Moreover, capecitabine-related toxicity may worsen due to drug-drug and drug-supplement interactions. Here we investigated factors responsible for severe HFS and diarrhoea presented by two patients, non-carriers of the recommended DPYD single nucleotide polymorphisms (SNPs) but carriers of other genetic variants suggested to increase the risk of capecitabine-related ADRs. Through careful therapy recognition, we demonstrated that, unbeknownst to the oncologists, the patients were taking folic acid during the treatment with capecitabine at a dosage higher than 2000 mg/m(2), which is the maximum tolerated dose when folate is administered. To resolve the ADRs, the therapy had to be drastically changed. In one case, dose reduction of capecitabine and discontinuation of lipid-lowering agents were carried out. In the other case, discontinuation of capecitabine and folic acid and capecitabine re-administration were performed after a month. Genetic and environmental factors should be considered good predictors of severe capecitabine-related toxicity. Medication reconciliation should be encouraged to avoid the harmful consequences of inappropriate treatments

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35’364 ternary pools of 52’746 siRNAs targeting 17’582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrP$^{Sc}$, and its cellular counterpart PrP$^{C}$, 40 genes affected selectively PrP$^{Sc}$. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrP$^{C}$. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions

“Touch microbiome” as a potential tool for forensic investigation: A pilot study

Human skin hosts a variety of microbes that can be transferred to surfaces (“touch microbiome”). These microorganisms can be considered as forensic markers similarly to “touch DNA”. With this pilot study, we wanted to evaluate the transferability and persistence of the “touch microbiome” on a surface after the deposition of a fingerprint and its exposure for 30 days at room temperature. Eleven volunteers were enrolled in the study. Skin microbiome samples were collected by swabbing the palm of their hands; additionally, donors were asked to touch a glass microscope slide to deposit their fingerprints, that were then swabbed. Both human and microbial DNA was isolated and quantified. Amelogenin locus and 16 human STRs were amplified, whereas the V4 region of 16 S rRNA gene was sequenced using Illumina MiSeq platform. STR profiles were successfully typed for 5 out of 22 “touch DNA” samples, while a microbiome profile was obtained for 20 out of 22 “touch microbiome” samples. Six skin core microbiome taxa were identified, as well as unique donor characterizing taxa. These unique taxa may have relevance for personal identification studies and may be useful to provide forensic intelligence information also when “touch DNA” fails. Additional future studies including greater datasets, additional time points and a greater number of surfaces may clarify the applicability of “touch microbiome” studies to real forensic contexts

An optogenetic intrabody tool for light-induced protein degradation in yeast

In recent years, intracellular antibodies (intrabodies) have been widely exploited as molecular tools both for basic research and potentially towards therapeutic applications. The general idea is to select target-specific recombinant antibodies, typically in the format of single chain variable fragments (scFvs) or nanobodies (Nb), stable in the intracellular reducing environment, and engineer them to interfere with the physiological or pathological activity of the target protein. Particularly, intrabodies are emerging as useful tools for knock-down/knock-out strategies at the protein level in order to manipulate the proteome of a cell with high rapidity, flexibility and specificity, without acting on genes or mRNAs (such as CRISPR, RNAi etc.). Moreover, since proteins inside the cells can exist in very different conformations and versions due the presence of post-translational modifications (PTM) and each form can play a very different role inside the cell, sometimes it is desirable to have a tool to eliminate just a particular conformation or PTM version of a given protein. In this scenario, the current screening strategies allow the selection of target-specific intrabodies, but one cannot be sure a priori to have selected intrinsically neutralizing antibodies. In this sense, the development of intrabodies able to guide the degradation of their target protein would be the solution as shown by the Suicide Intrabody Technology (SIT). However, SIT do not offer a sufficient orthogonal tool for these purposes, due to the fact that here the degradation of the target, through the ubiquitin-dependent pathway, works upon administration of the TNFα, a chemical inductor with different functions inside the cell. The goal of this master thesis was the development of a light-dependent intrabody-based tool (optobodies) for a rapid, reversible, selective and orthogonal regulation of the proteostasis inside a cell, with high temporal and, potentially, spatial resolution. The idea of the project was to establish a proof-of-concept about the possibility to control the lifetime of potentially every protein simply through a light stimulus, avoiding non-specific effects. For these purposes, a fusion protein was created, in which an intrabody was bound to a photoinducible degron module, composed by a degron unit and a light-sensitive domain, called LOV domain: this last unit masks the degron, that can be activated through a conformational switch induced by light. The tool was tasted and validated in yeast, as a robust and simple eukaryotic cellular model, optimal for such a type of proof-of-principle experiments. The effectiveness of the intrabody-LOV domain construct to control the light-induced selective target protein degradation was evaluated for three different intrabodies previously selected: the single-chain intrabody scFv58F, selected against an acetylated lysine on the histone H3 (H3K9Ac), and two nanobodies NbSyn2 and NbSyn87, able to target the monomeric form of the protein α-synuclein. The optimal illumination conditions were set up and the efficiency of the system was evaluated by western blots, to quantify the degradation of the targets, and trying to induct visible phenotypes in living yeast cells. The data obtained clearly show that light-induced protein degradation was achieved for two of the three intrabodies tested in this proof-of-concept

ON THE DAMAGE TOLERANCE OF C/C-SIC COMPOSITE HOT STRUCTURES

Thanks to their excellent structural performance at high temperature, the Ceramic‐Matrix Composites (CMCs) are good candidates for light‐weight structures in high temperature applications, such as applications to re-entry vehicles. Beyond the well-known high temperature resistance, the CMCs do also show good damage tolerance properties, making them suitable for applications to large primary structures. Unfortunately, the production at the industrial level is currently at an embryonic stage due to the criticalities in the involved manufacturing processes which do not allow to produce defects free components. During the design phase, the presence of these manufacturing related defects are taken into account by introducing knock-down factors reducing the mechanical properties. However, this approach can be highly conservative and can strongly limit the intrinsic advantages of using such materials. Moreover, even if suitable non-destructive techniques (such as tomography) can be adopted to identify the manufacturing defects, still uncertainties exist about these defects evolution and, above all, about their influence on the structural performance (in term of stiffness and strength) of real components. The adoption of suitable material and fracture mechanics numerical models, could help to correctly predict the stiffness and strength characteristics of CMCs at coupon and subcomponent level. Which such advanced numerical models the manufacturing defects and their influence on the structural performance could be taken into account leading to a strong reduction of the currently adopted knockdown factors. The present work aims to investigate the damage tolerance of a C/C-SiC hot structure acting as aerodynamic control surface of a reusable re-entry vehicle. These vehicles are exposed to severe environmental conditions when re-entering Earth atmosphere. Indeed, the highest loaded areas such as nose, leading edges and control surfaces can experience temperatures up to 1650°C. Since the structural integrity of a re-entry vehicle needs to be guaranteed during all the mission phases and for multiple missions, a damage tolerant reliable design is required. Starting from the validation at coupon level of the material and fracture mechanics numerical models, by means of experimental data available in literature, a full parametric Finite Element model has been developed. The proposed parametric model is able to consider any delamination in any planar and thickness location all along the body flap domain. In order to reduce the computational costs, the entire body flap was modelled by means of layered shell elements, while layered solid elements have been used in the delaminated area. Hence, to connect the global coarse model and the local refined model, a global-local approach has been implemented. Finally, a sensitivity analysis has been performed finalised to assess the influence of location (in plane and trough the thickness) and dimension (radius) of a circular delamination on the damage tolerance of the investigated structure

A straight forward procedure to extract the volcanic cloud top height as a Plume Elevation Model (PEM) from already Orthorectified Landsat 8 images. Application to the 26 October 2013 Mt Etna eruption (Italy) and cross comparison with Lagrangian trajectories, Modis and Iasi data.

International audienc

Volcanic Cloud Top Height Estimation Using the Plume Elevation Model Procedure Applied to Orthorectified Landsat 8 Data. Test Case: 26 October 2013 Mt. Etna Eruption

International audienceIn this study, we present a method for extracting the volcanic cloud top height (VCTH) as a plume elevation model (PEM) from orthorectified Landsat 8 data (Level 1). A similar methodology was previously applied to raw Landsat-8 data (Level 0). But level 0 data are not the standard product provided by the National Aeronautics and Space Administration (NASA)/United States Geological Survey (USGS). Level 0 data are available only on demand and consist on 14 data stripes multiplied by the number of multispectral bands. The standard product for Landsat 8 is the ortho image, available free of charge for end-users. Therefore, there is the need to adapt our previous methodology to Level 1 Landsat data. The advantages of using the standard Landsat products instead of raw data mainly include the fast -ready to use- availability of the data and free access to registered users, which is of major importance during volcanic crises. In this study, we adapt the PEM methodology to the standard Landsat-8 products, with the aim of simplifying the procedure for routine monitoring, offering an opportunity to produce PEM maps. In this study, we present the method. Our approach is applied to the 26 October 2013 Mt. Etna episodes comparing results independent VCTH measures from the spinning enhanced visible and infrared imager (SEVIRI) and the moderate resolution imaging spectroradiometer (MODIS)

Evaluations of Muscular Strength, Ability to Balance and Health Status in Prisoners during COVID-19

Recent events in prisons during the COVID-19 pandemic showed how the health situation and overcrowding in prisons are a source of high risk to the health and physical and mental wellbeing of the prison population and how this has become an important medical problem. The original purpose of this study, which was initially planned to last 6 months, was to examine the effects of a training program on cardio-respiratory capacity, resistance to dynamic strength of the upper and lower body and muscle mass. Following the COVID-19 pandemic, the purpose was subsequently modified by highlighting whether and which deficiencies occurred as a result of the absence of physical activity. Forty adult men between 35 and 55 years of age with more than 1 year of detention were selected and randomly divided into two groups: the experimental group and control group. The fitness training protocol of the experimental group consisted of three weekly sessions lasting 90 min, while control group subjects followed a walk of 30–60 min three days a week without running or resistance training. The unpaired and paired t-tests revealed significant effects of both health status and fitness level (p < 0.05; p < 0.01) on group training. The results of this research show that prisoners can improve their fitness and health through participation in physical education programs. This conclusion is especially important for prisoners who have to serve very long prison sentences and who are at great risk of showing poor physical condition levels