Nanoparticulate architecture of protein-based artificial viruses is supported by protein DNA interactions

Aim & Methods: We have produced two chimerical peptides of 10.2 kDa, each contain four biologically active domains, which act as building blocks of protein-based nonviral vehicles for gene therapy. In solution, these peptides tend to aggregate as amorphous clusters of more than 1000 nm, while the presence of DNA promotes their architectonic reorganization as mechanically stable nanometric spherical entities of approximately 80 nm that penetrate mammalian cells through arginine–glycine–aspartic acid cell-binding domains and promote significant transgene expression levels. Results & Conclusion: The structural analysis of the protein in these hybrid nanoparticles indicates a molecular conformation with predominance of D-helix and the absence of cross-molecular, E-sheet-supported protein interactions. The nanoscale organizing forces generated by DNA–protein interactions can then be observed as a potentially tunable, critical factor in the design of protein-only based artificial viruses for gene therapy.Agencia Nacional de Investigación e Innovació

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Development and characterization of artificial viruses for gene therapy

Els riscos biològics associats a la teràpia gènica viral limiten el ple desenvolupament de vectors virals i plantegen importants problemes a la seva incorporació en assajos clínics. La teràpia gènica no viral representa una alternativa segura als virus naturals per al lliurament dirigida de gens a cèl·lules, tot i els baixos nivells d'expressió gènica obtinguts que és l’obstacle principal per la seva aplicació terapèutica. Els diferents tipus de vectors no virals que s'han desenvolupat fins ara, inclouen els basats en liposomes, dendrímers o proteïnes. Recentment, el concepte de "virus artificial” s'ha proposat per descriure nanocomplexes per al lliurament de gens que imiten les funcions virals pertinents per a la captació del gen i el tràfic intracel·lular. Entre ells, els basats en proteïnes i construïts a través de principis modulars permeten la incorporació, en un únic polipèptid, de diferents proteïnes o dominis de proteïnes amb funcions similars a virus, és a dir, la unió i la condensació a ADN, la unió al receptor, la internalització, l’escapament endosomal, la localització nuclear i l’alliberament del material transportat. Hem desenvolupat una sèrie de vehicles proteics modulars formades per diferents dominis funcionals que són capaços d'entrar en les cèl·lules a través de la unió un receptor específic i promoure nivells importants de l'expressió gènica. En aquesta tesi s'analitza aquest enfocament amb dos articles de revisió i es demostra amb tres treballs originals.The biological risks associated to viral gene therapy limit the full development of viral vectors and pose major concerns to their incorporation into clinical trials. Non-viral gene therapy represents a safe alternative to natural viruses for cell targeted gene delivery, although the low gene expression levels achieved by non-viral vectors are a main obstacle for their therapeutic application. Different types of non-viral vectors have been developed up to date, including those based in liposomes, dendrimers or proteins. Recently, the ‘Artificial virus’ concept has been proposed to describe nanocomplexes for gene delivery that mimic the viral functions relevant to gene uptake and intracellular trafficking. Among them, those based on proteins and constructed through modular principles allow the incorporation, in a single polypeptide, of different proteins or protein domains with virus-like functions, namely DNA binding and condensation, receptor binding, internalization, endosomal escape, nuclear targeting and uncoating. We have developed a series of protein-only modular vehicles composed by different functional domains that are able to enter cells through specific receptor binding and promotes important levels of transgene expression. In this thesis this approach is discussed with two review articles and demonstrated with three original papers

Synchrotron radiation circular dichroism spectroscopy reveals structural divergences in HDL-bound apoA-I variants

Abstract Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) provides cardiovascular protection. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to analyze the dynamic solution structure of the apoA-I protein in the apo- and HDL-states and the protein structure conversion in HDL formation. Wild-type apoA-I protein was compared to human variants that either are protective (R173C, Milano) or lead to increased risk for ischaemic heart disease (A164S). Comparable secondary structure distributions in the HDL particles, including significant levels of beta strand/turn, were observed. ApoA-I Milano in HDL displayed larger size heterogeneity, increased protein flexibility, and an altered lipid-binding profile, whereas the apoA-I A164S in HDL showed decrease thermal stability, potentially linking the intrinsic HDL propensities of the variants to disease risk

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose

Dual actions of apolipoprotein A-I on glucose-stimulated insulin secretion and insulin-independent peripheral tissue glucose uptake lead to increased heart and skeletal muscle glucose disposal

Apolipoprotein A-I (apoA-I) of HDL is central to the transport of cholesterol in circulation. ApoA-I also provides glucose control with described in vitro effects of apoA-I on β-cell insulin secretion and muscle glucose uptake. In addition, apoA-I injections in insulin-resistant diet-induced obese (DIO) mice lead to increased glucose-stimulated insulin secretion (GSIS) and peripheral tissue glucose uptake. However, the relative contribution of apoA-I as an enhancer of GSIS in vivo and as a direct stimulator of insulin-independent glucose uptake is not known. Here, DIO mice with instant and transient blockade of insulin secretion were used in glucose tolerance tests and in positron emission tomography analyses. Data demonstrate that apoA-I to an equal extent enhances GSIS and acts as peripheral tissue activator of insulin-independent glucose uptake and verify skeletal muscle as an apoA-I target tissue. Intriguingly, our analyses also identify the heart as an important target tissue for the apoA-I-stimulated glucose uptake, with potential implications in diabetic cardiomyopathy. Explorations of apoA-I as a novel antidiabetic drug should extend to treatments of diabetic cardiomyopathy and other cardiovascular diseases in patients with diabetes

Comparative evaluation of stabilizers for plasticized polyvinyl chloride

Translated from Bulgarian (Khim. Ind. 1988 v. 60(8) p. 365-367)Available from British Library Document Supply Centre- DSC:9022.0601(BISI-EM-Trans--341)T / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Microbial factories for recombinant pharmaceuticals

Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative wa