Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedureThis work has been supported by grants PID 2020-119627GB-I00 and DTS21/00134 from Ministerio Español de Ciencia e Innovación (Spain) from to MY-M. BB is supported by predoctoral industrial contract IND2019_BMD-17100 and JM by a INV-CAM-03 Yo Investigo contract, both from Comunidad Autónoma de Madrid. Work was partially funded from the European Union’s Horizon 2020 research and innovation program under grant agreements No. 951768 (project MARVEL

Chemical perturbation of oncogenic protein folding: from the prediction of locally unstable structures to the design of disruptors of Hsp90-Client interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein‐90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co‐chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c‐Abl, c‐Src, Cdk4, B‐Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90‐system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

A Statistical Analysis of the Trading Agent Competition 2001

We present a statistical analysis on the data from the Second Trading Agent Competition (TAC01). Our goal is to study the effectiveness of this competition as a testbed for the evaluation of trading strategies in artificial markets. Our results suggest that in TAC01 no agent performed significantly better than all the others, from a pure statistical viewpoint. Instead, it reveals groups of agents which performed significantly better than others. Thus, our results suggest that although this competition may not give a quantitative evaluation of the agents' trading strategies, it can still provide some useful qualitative evaluation

Indicatori e modelli per lo sviluppo sostenibile del territorio e la valorizzazione del paesaggio. Ambito metropolitano: area nord-est di Milano

Le aree rurali in ambito metropolitano presentano caratteristiche e problematiche peculiari rispetto alle altre tipologie di aree rurali, in ragione loro localizzazione prossima alla citt\ue0\ua0. Le caratteristiche e le valenze di tali aree possono essere descritte in maniera sintetica e significativa attraverso l'utilizzo e la definizione di alcuni indicatori. Ci\uf2 anche al fine di effettuare confronti temporali e spaziali e di valutare gli effetti delle politiche sul territorio. Il presente lavoro \ue8 suddiviso in due momenti distinti. In un primo tempo sono stati individuati gli indicatori e sono stati raccolti i dati riguardanti il territorio in esame. In un secondo momento si \ue8 rivolta maggiore attenzione al livello comunale per individuare alternative spaziali allo sviluppo urbano e valutare quella in grado di rispondere meglio all'obiettvo generale. in tal senso viene utilizzato un approccio basato sull'integrazione tra metodi di analisi e valutazione delle risorse basati sull'impiego dello strumento GIS (EPP) con metodi di analisi multicriteriali basati sull'AHP (Analytic Hierarchy Process

A new model for 3D graphical rendering

One of the most important tasks of a traditional 3D Rendering engine is the projection on the image plane of geometrical structures (such as triangles or lines). This operation takes place in the middle of the rendering pipeline, between the vertex shader and the fragment shader: its aim is just that of creating fragment data from vertex data. The solution of the projection problem is necessarily bound to the solution of a great number of systems of equations, where the complexity of the equations is in general related to the properties of the geometrical structures. To make this process fast, the most adopted solution is that of using linear models, so that the systems become linear and the module gets the simplest implementation. Unfortunately, linear models have some limitations: the solution is to use approximation, but to get good models they are necessary a lot of linear structures, in particular a lot of triangles; modern 3D Rendering Engines may automate the process of converting non linear models in triangles, but this does not reduce the occupation of memory and doesn’t eliminate linear approximation. In this article I consider a non linear model (the Lembo model) for geometrical structures in a 3D rendering engine: firstly I show the properties of the model; then I show an efficient algorithm to solve the projection problem directly on the model equations

Acid Catalyzed Formation of C–C and C–S Bonds via Excited State Proton Transfer

The behavior of 2-naphthol and 7-bromo-2-naphthol as organic photoacids are exploited in organic synthesis for the preparation of benzyl sulfides (using a trichloroacetimidate derivative as the starting substrate) and polycyclic amines via acid catalyzed condensation of 1,2,3,4-tetrahydroisoquinoline with aldehydes