Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa

[EN] Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4- HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC)= 0.820, P < 0.05] and percentages of caspase 3- and 4-HNEpositive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alphapositive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the importance of considering different markers of oxidative stress.S

Weld kinematics of synrift salt during basement-involved extension and subsequent inversion: Results from analog models

Scaled analog models based on extensional basins with synrift salt show how basement topography exerts a control factor on weld kinematics during the extension and inversion phases. In the case of basement-involved extension, syn-rift salt thickness differences may lead to variable degrees of extensional decoupling between basement topography and overburden, which in turn have a strong impact on the development of salt structures. With ongoing extension and after welding, the basin kinematics evolves toward a coupled deformation style. The basin architecture of our experimental results record the halokinetic activity related to growing diapirs and the timing of weld formation during extension. Moreover, the structures that result from any subsequent inversion of these basins strongly depends on the inherited welds and salt structures. While those basins are uplifted, the main contractional deformation during inversion is absorbed by the pre-existing salt structures, whose are squeezed developing secondary welds that often evolve into thrust welds. The analysis of our analog models shows that shortening of diapirs is favored by: 1) basement topography changes that induce reactivation of primary welds as thrust welds; 2) reactivation of the salt unit as a contractional detachment; and 3) synkinematic sedimentation during basin inversion. Finally in this article we also compare two natural examples from the southern North Sea that highlight deformation patterns very similar to those observed in our analog models

Paleogeographic and Sedimentary evolution of the South-Pyrenean Foreland basin

During the Paleogene and Neogene the NE Iberian plate underwent significant paleogeographic changes driven by the Iberian and European plate collision and the resulting formation of the Pyrenean orogen and its corresponding foreland basin. Shortening resulted in the advance of the orogenic wedge, emplacement of allochthonous units, and progressive basin partitioning. Sediment transfer systems reacted to the evolving paleogeographic scenario, shifting from forebulge to foredeep and wedge-top settings. Critical reorganizations included successive shifts from open to close drainage conditions, which had an strong impact on accommodation, and the stratigraphic architecture of the basin infill, overfill and later erosion. The aim of this work is to synthesize the paleogeographic and sedimentary evolution of the south-pyrenean foreland, with emphasis on the reconstruction of sediment routing, the evaluation of sedimentation rate trends, the timing of sedimentary shifts and the analysis of their causes. Stratigraphic data are compiled in a comprehensive magnetostratigraphy-based chronostratigraphic framework. Besides, sedimentary and structural data are put together to produce a series of palinspastically restored paleogeographic maps, which reflect five key stages in the evolution of the region. These stages include: 1) the Late Cretaceous tectonic inversion of the extended Iberian margin; 2) the Early Eocene formation of the southern Pyrenean foredeep; 3) the Middle Eocene widening and overfilling; 4) the late Eocene shift into an internal drainage; and 5) the Late Miocene drainage opening and erosion. In the light of these results, the variable role of tectonics, climate and eustacy at different time scales are discussed

El aprovechamiento de la campiña entre Teba y Ardales (Málaga) por los agricultores del Neolítico: el caso del Cerro de la Higuera

Durante la construcción del parque eólico “La Higuera”, en los términos municipales de Teba y Ardales (Málaga) se localizaron, al aire libre, productos arqueológicos prehistóricos que han permitido analizar lo que fue un asentamiento de agricultores del Neolítico

Further validation and psychometric properties of the Spanish adaptation of the Genetic Counseling Outcome Scale

Evaluation of clinical genetic services is challenging due to the nature of their interventions. The Genetic Counseling Outcome Scale (GCOS-24), a patient-reported outcome measure, was developed to measure empowerment, an important patient-reported outcome from genetic counseling. Previously, we translated and adapted GCOS-24 for use in Spain, but neither test–retest reliability nor structural and construct validity were assessed at that time. In the present study, we set out to test the reliability and validity of the Spanish adaptation of the GCOS-24 against already validated Spanish language measures of satisfaction with life, anxiety, and health locus of control. 880 patients/families who attended the genetics clinic were invited to participate in a online survey. 201 participants (23%) completed the four questionnaires at the first timepoint, and 59 of these (29%) completed GCOS-24 again the second timepoint, 2–4 weeks later. Test–retest reliability was confirmed, with no significant differences between responses to GCOS-24 at the first and second timepoints and good internal consistency. Convergent validity was confirmed between GCOS-24 and measures of satisfaction with life and anxiety but not with measures of health locus of control. For the structural and construct validation, an exploratory factor analysis was performed. The resulting factorial structure of GCOS-24 consists of 6 factors that accumulate 68% of the variance shared by the 21 items that remained in the model. We applied the factor structure of the three validated measures to the available data and analyzed the correlation between factors of GCOS-24 and the other scales. The results showed significant and consistent correlation with factors of the satisfaction with life and anxiety scales but no significant correlation with internal health locus of control. The use of the Spanish adaptation of GCOS-24 in other genetic clinics in Spain will help to validate it further. This study contributes to the international validation of GCOS-24 to evaluate the quality of genetic counseling in Europe

Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes

[EN] The reduced lifespan of cryopreserved spermatozoa in the mare reproductive tract has been attributed to both capacitative and apoptotic changes. However, there is a lack of studies investigating both phenomena simultaneously. In order to improve our knowledge in this particular point, we studied in raw and frozen-thawed samples apoptotic and capacitative markers using a wide battery of test based in flow cytometry. Apoptotic markers evaluated were caspase 3 activity, externalization of phosphatidylserine (PS), and mitochondrial membrane potential. Markers of changes resembling capacitation were membrane fluidity, tyrosine phosphorylation, and intracellular sodium. Conventional and computational flow cytometry using nonlinear dimensionally reduction techniques (t-distributed stochastic neighbor embedding (t-SNE)) and automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE) were used. Most of the changes induced by cryopreservation were apoptotic, with increase in caspase 3 activation (P < 0.01), PS translocation to the outer membrane (P < 0.001), loss of mitochondrial membrane potential (P < 0.05), and increase in intracellular Na+ (P < 0.01). Average values of markers of capacitative changes were not affected by cryopreservation; however, the analysis of the phenotype of individual spermatozoa using computational flow cytometry revealed the presence of subpopulations of spermatozoa experiencing capacitative changes. For the first time advanced computational techniques were applied to the analysis of spermatozoa, and these techniques were able to disclose relevant information of the ejaculate that remained hidden using conventional flow cytometry.SIThe authors received financial support for this study from the Ministerio de Economía y Competitividad-FEDER, Madrid, Spain, grant AGL2013-43211-R, Junta de Extremadura-FEDER (GR 15029). PMM is supported by a pre-doctoral grant from the Ministerio de Educación, Cultura y Deporte, Madrid Spain FPU13/03991. COF is supported by a post-doctoral grant from the Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa

[EN] We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.SIThe authors received financial support for this study from the Ministerio de Economía y Competitividad-FEDER, Madrid, Spain, grants AGL2013-43211-R, AGL2017-83149-R Junta de Extremadura-FEDER (European Regional Development Fund) (GR 18008 and IB16030). PMM is supported by a pre-doctoral grant from the Ministerio de Educación, Cultura y Deporte, Madrid Spain FPU13/03991. COF is supported by a post-doctoral grant from the Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671, JMO-R holds a PhD grant from Valhondo Calaaf Foundation, Cáceres Spain

MVA-CoV2-S vaccine candidate confers full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice

The protective efficacy of vaccines against SARS-CoV-2 infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe COVID-19 disease, we report a detailed spatiotemporal description of the SARS-CoV-2 infection and replication in different areas of the brain. Remarkably, SARS-CoV-2 brain replication occurs primarily in neurons, producing important neuropathological alterations such as neuronal loss, incipient signs of neuroinflammation, and vascular damage in SARS-CoV-2 infected mice. Notably, one or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. To our knowledge, this is the first study of a COVID-19 vaccine candidate showing 100% efficacy against SARS-CoV-2 brain infection and damage, reinforcing the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19, worth to move forward into clinical trials.The authors thank the Centro de Investigación en Sanidad Animal (CISA)-Instituto Nacional de Investigaciones Agrarias (INIA-CSIC) (Valdeolmos, Madrid, Spain) for the BSL-3 facilities. SARS-CoV-2 MAD6 virus isolate was kindly provided by José M. Honrubia and Dr. Luis Enjuanes (CNB-CSIC, Madrid, Spain). We also thank to Dr. Konstantin L. Levitsky for excellent technical assistance with the confocal acquisition. We thank the Spanish Research Council (CSIC) and the Spanish Ministry of Science and Innovation (MICINN) for continuous support. This research was supported by MCIN/Spanish Research Agency (AEI)/ 10.13039/501100011033 grants: PID2019-105995RB-I00 (J.T.-A. and J.V.), PID2020- 114481RB-I00 (J.G.-A. and M.E.), and PID2019-106410RB-I00 (J.L.-B.). Moreover, this research work was also funded by Red TerCel ISCIII, RD16/0011/0025 (J.T.-A.); Consejería de Salud y Familias, Junta de Andalucía Grant, PECOVID-0078-2020 (R.R.-L. and J.V.); Fondo COVID-19 grant COV20/00151 [Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII)], Fondo Supera COVID-19 (Crue Universidades-Banco Santander) grant and CSIC grant 202120E079 (J.G.-A.); and CSIC grant 2020E84, La CaixaImpulse grant CF01-00008, Ferrovial and MAPFRE donations (M.E.). Additionally, we have also funding from the European Commission-NextGenerationEU, through CSIC's Global Health Platform (PTI Salud Global) (J.G.-A. and M.E.) and the European Research Council (ERC Advanced Grant PRJ201502629) (J.L.-B.).N

Full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice conferred by MVA-CoV2-S vaccine candidate

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.We thank the CSIC and the Spanish Ministry of Science and Innovation for continuous support. This research was supported by the Spanish Ministry of Science and Innovation/Spanish Research Agency/10.13039/501100011033 grant nos. PID2019-105995RB-I00 (J.J.T.-A. and J.V.), PID2020-114481RB-I00 (J.G.-A. and M.E.), RTI2018-096629-B-I00 (A.P.) and PID2019-106410RB-I00 (J.L.-B.). Moreover, this research was also funded by Red TerCel ISCIII (no. RD16/0011/0025 to J.J.T.-A.), Consejería de Economía, Conocimiento, Empresas y Universidad US-1380891 (to J.J.T.-A. and J.V.), Consejería de Salud y Familias, Junta de Andalucía grant no. PECOVID-0078-2020 (to R.R.-L. and J.V.), Consejería de Educación y Deporte, Junta de Andalucía grant no. PY20_01312 (to A.P.), Fondo COVID-19 grant no. COV20/00151 (Spanish Health Ministry, Instituto de Salud Carlos III), Fondo Supera COVID-19 (Crue Universidades-Banco Santander) grant and CSIC grant no. 202120E079 (J.G.-A.), CSIC grant no. 2020E84, La CaixaImpulse grant no. CF01-00008 and Ferrovial and MAPFRE donations (to M.E.). Additionally, we received funding from the European Commission-NextGenerationEU, through the CSIC’s Global Health Platform (PTI Salud Global) (to J.G.-A. and M.E.) and the European Research Council (ERC Advanced grant no. PRJ201502629) (to J.L.-B.). J.G.-A. and M.E. also acknowledge financial support from the Spanish State Research Agency (no. AEI/10.13039/501100011033) through the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (nos. SEV-2013-0347 and SEV-2017-0712). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Peer reviewe

10 Myr evolution of sedimentation rates in a deep marine to non-marine foreland basin system: Tectonic and sedimentary controls (Eocene, Tremp–Jaca Basin, Southern Pyrenees, NE Spain)

The propagation of the deformation front in foreland systems is typically accompanied by the incorporation of parts of the basin into wedge-top piggy-back basins, this process is likely producing considerable changes to sedimentation rates (SR). Here we investigate the spatial-temporal evolution of SR for the Tremp–Jaca Basin in the Southern Pyrenees during its evolution from a wedge-top, foreredeep, forebulge configuration to a wedge-top stage. SR were controlled by a series of tectonic structures that influenced subsidence distribution and modified the sediment dispersal patterns. We compare the decompacted SR calculated from 12 magnetostratigraphic sections located throughout the Tremp–Jaca Basin represent the full range of depositional environment and times. While the derived long-term SR range between 9.0 and 84.5 cm/kyr, compiled data at the scale of magnetozones (0.1–2.5 Myr) yield SR that range from 3.0 to 170 cm/kyr. From this analysis, three main types of depocenter are recognized: a regional depocenter in the foredeep depozone; depocenters related to both regional subsidence and salt tectonics in the wedge-top depozone; and a depocenter related to clastic shelf building showing transgressive and regressive trends with graded and non-graded episodes. From the evolution of SR we distinguish two stages. The Lutetian Stage (from 49.1–41.2 Ma) portrays a compartmentalized basin characterized by variable SR in dominantly underfilled accommodation areas. The markedly different advance of the deformation front between the Central and Western Pyrenees resulted in a complex distribution of the foreland depozones during this stage. The Bartonian–Priabonian Stage (41.2–36.9 Ma) represents the integration of the whole basin into the wedge-top, showing a generalized reduction of SR in a mostly overfilled relatively uniform basin. The stacking of basement units in the hinterland during the whole period produced unusually high SR in the wedge-top depozoneAgència de Gestió d'Ajuts Universitaris i de Recerca, Grant/Award Number: 2017SGR596; Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: BES-2015-073302 and CGL2014-55900-P; Swiss National Science Foundation, Grant/Award Number: 200020_18201