Structural Analysis of Human Respiratory Syncytial Virus P Protein: Identification of Intrinsically Disordered Domains

Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity

Background and Aims The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help monitoring seed deterioration. Methods Dry seeds were subjected to artificial aging and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (Random Amplified Polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin Ciocalteu reagent method. Electron Paramagnetic Resonance (EPR) provided profiles of free radicals. Quantitative RealTime-Polymerase Chain Reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (Type 2 Metallothionein) and SOD (Superoxide Dismutase). A modified QRT-PCR protocol was used to determine telomere length. Key Results The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial aging. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterised by a lower antioxidant specific activity. Significant up-regulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. Conclusions Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species

Anti-inflammatory and antioxidant nanostructured cellulose membranes loaded with phenolic-based ionic liquids for cutaneous application

The utilization of natural compounds, such as phenolic acids and biopolymers, in the healthcare domain is gaining increasing attention. In this study, bacterial nanocellulose (BC) membranes were loaded with ionic liquids (ILs) based on phenolic acids. These ionic compounds, with improved solubility and bioavailability, were prepared by combining the cholinium cation with anions derived from caffeic, ellagic and gallic acids. The obtained BC-ILs membranes were homogeneous, conformable and their swelling ability agreed with the solubility of each IL. These membranes revealed a controlled ILs dissolution rate in the wet state and high antioxidant activity. In vitro assays performed with Raw 264.7 macrophages and HaCaT keratinocytes revealed that these novel BC-ILs membranes are non-cytotoxic and present relevant anti-inflammatory properties. Diffusion studies with Hanson vertical diffusion cells showed a prolonged release profile of the ILs from the BC membranes. Thus, this work, successfully demonstrates the potential of BC-ILs membranes for skin treatment.publishe

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.CNPqINCTVFAPESPUS

Manejo de la inmunosupresión en pacientes trasplantados de riñón con COVID19. Estudio multicéntrico nacional derivado del registro COVID de la Sociedad Española de Nefrología

Introduction: SARS CoV2 infection has had a major impact on renal transplant patients with a high mortality in the first months of the pandemic. Intentional reduction of immunosuppressive therapy has been postulated as one of the cornerstone in the management of the infection in the absence of targeted antiviral treatment. This has been modified according to the patient`s clinical situation and its effect on renal function or anti-HLA antibodies in the medium term has not been evaluated.Objectives: Evaluate the management of immunosuppressive therapy made during SARS-CoV2 infection, as well as renal function and anti-HLA antibodies in kidney transplant patients 6 months after COVID19 diagnosis.Material and methods: Retrospective, national multicentre, retrospective study (30 centres) of kidney transplant recipients with COVID19 from 01/02/20 to 31/12/20. Clinical variables were collected from medical records and included in an anonymised database. SPSS statistical software was used for data analysis.Results: renal transplant recipients with COVID19 were included (62.6% male), with a mean age of 57.5 years. The predominant immunosuppressive treatment prior to COVID19 was triple therapy with prednisone, tacrolimus and mycophenolic acid (54.6%) followed by m-TOR inhibitor regimens (18.6%). After diagnosis of infection, mycophenolic acid was discontinued in 73.8% of patients, m-TOR inhibitor in 41.4%, tacrolimus in 10.5% and cyclosporin A in 10%. In turn, 26.9% received dexamethasone and 50.9% were started on or had their baseline prednisone dose increased. Mean creatinine before diagnosis of COVID19, at diagnosis and at 6 months was: 1.7 +/- 0.8, 2.1 +/- 1.2 and 1.8 +/- 1 mg/dl respectively (p < 0.001). 56.9% of the patients (N = 350) were monitored for anti-HLA antibodies. 94% (N = 329) had no anti-HLA changes, while 6% (N = 21) had positive anti-HLA antibodies. Among the patients with donor-specific antibodies post-COVID19 (N = 9), 7 patients (3.1%) had one immunosuppressant discontinued (5 patients had mycophenolic acid and 2 had tacrolimus), 1 patient had both immunosuppressants discontinued (3.4%) and 1 patient had no change in immunosuppression (1.1%), these differences were not significant.Conclusions: The management of immunosuppressive therapy after diagnosis of COVID19 was primarily based on discontinuation of mycophenolic acid with very discrete reductions or discontinuations of calcineurin inhibitors. This immunosuppression management did not influence renal function or changes in anti-HLA antibodies 6 months after diagnosis

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

An asymmetry detected in the disk of Kappa CMa with the AMBER/VLTI

International audienceAims. We study the geometry and kinematics of the circumstellar environment of the Be star Kappa CMa in the Br gamma emission line and its nearby continuum. Methods. We use the VLTI/AMBER instrument operating in the K band which provides a spatial resolution of about 6 mas with a spectral resolution of 1500 to study the kinematics within the disk and to infer its rotation law. In order to obtain more kinematical constraints we also use an high spectral resolution Pa beta line profile obtain in December 2005 at the Observatorio do Pico do Dios, Brazil and we compile V/R line profile variations and spectral energy distribution data points from the literature. Results. Using differential visibilities and differential phases across the Br gamma line we detect an asymmetry in the disk. Moreover, we found that kappa CMa seems difficult to fit within the classical scenario for Be stars, illustrated recently by alpha Arae observations, i.e. a fast rotating B star close to its breakup velocity surrounded by a Keplerian circumstellar disk with an enhanced polar wind. Finally we discuss the possibility for kappa CMa to be a critical rotator with a Keplerian rotating disk and try to see if the detected asymmetry can be interpreted within the "one-armed" viscous disk framework