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12 Pág., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants., This research work was funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC´s Global Health Platform (PTI Salud Global), grants SGL 2103051 (to MS and FS) and SGL2103053 (to MM-A). It was further supported by the Spanish Ministry of Science and Innovation, grant PID2020-113184RB-C21 (to FS and MS) and by grant S018/BAA-4370 (co-financed by the Autonomous Community of Madrid and EC FEDER funds, to FS). MP was the recipient of a scholarship from the CSIC´s JAE Intro program.

Additional file 5: Fig. S3. Boxplots showing the distribution of the morphometric variables of the four subpopulations along with mean comparison P-values obtained by the t-test.

Additional file 2: Table S2. Mean values (± S.D.) of each morphometric parameter corresponding to different SPs from bull sperm head morphometry found in sperm migrated or not migrated by thermotaxis.

Additional file 1: Table S1. Eigenvalues of each parameter in the three PCs for bull sperm head morphometry found in sperm migrated or not migrated by thermotaxis.

Additional file 3: Fig. S1. Density plots showing the distribution of the 8 morphometric variables in both migrated and not-migrated spermatozoa after thermotaxis.

Additional file 4: Fig. S2. Distribution of sperm subpopulations according to their PC values.

Additional file 1: Table S1. Details of primers used for reverse transcription–quantitative polymerase chain reaction.

14 Pág., The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P., This research was funded by the INIA project RTA2017-00034-00-00. Part of this research received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 677353 IMAGE.

Additional file 2: Table S2. Top 20 miRNA in EVs from OF and UF.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

Departamento de Reproducción animal, Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies., This work was supported by the Spanish Ministry of Science and Innovation (AGL-2015-70140-R, PID2019-111641RB-I00, and RTI2018-093548-B-I00); Y.N.C. was supported by Secretaría de Educación Superior, Ciencia y Tecnología e Innovación (SENESCYT-Ecuador); São Paulo Research Foundation, Brazil (FAPESP; #2017/20339-3, #2014/22887-0, and #2019/04981-2); and National Council for Scientific and Technological Development—CNPq, Brazil (grant number #420152/2018-0). The Authors are members of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit).

Additional file 1: Summary of sequencing statistics of each sample.

Additional file 2: Mean methylation levels of genomic elements.