In vitro and in vivo development of mice morulae after storage in non-frozen conditions

Background Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment. Methods Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR. Results In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C. Conclusions Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.This work was performed as part of, and financed by, Project AGL2004-00332. JDH received a Ph D. grant from the INIA (Ministry of Science and Innovation). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its unit of Information Resources for Research (URICI).Peer Reviewe

A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Individual proteins chemically labeled with fluorescent dyes can be localized and tracked in real-time experiments in order to get insights about the site and molecular mechanism of action. Here, we have adapted a protocol that was originally developed for two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) applications, to label proteins with CyDye fluors for single-molecule internalization assays in living cells. This “minimal labeling” method offers a number of advantages including specificity and known stoichiometry, simplicity, high reproducibility, and sensitivity and allows multiplexing while minimizing perturbations of the biological system. Moreover, since only a single lysine (Lys) residue per protein molecule is labeled, this method is also quantitative. To validate experimentally our protocol, we carried out the fluorescent labeling of IBB1, a major soybean protease isoinhibitor of the Bowman-Birk family that is currently being investigated as colorectal chemopreventive agent. Then, we analyzed the in vivo internalization dynamics of the labeled IBB1 protein in human colorectal adenocarcinoma HT29 cells

Shallow-impurity states of semiconductor Fibonacci superlattices

A theoretical study of shallow-donor states of GaAs-(Ga,Al)As semiconducting quasiperiodic Fibonacci superlattices is presented. The impurity states are calculated using different variational methods within the parabolic-band model and effective-mass approximation. We deal with periodic superlattices having a Fibonacci sequence of GaAs and (Ga,Al)As layers as unit cells, the size of these sequences being of increasing order. The binding energy and effective mass associated with the Is-like shallow-impurity states of these systems show a dependence on the donor position in the superlattices, which reflects the self-similarity and quasiperiodicity of the Fibonacci superlattices. We present a detailed explanation of the Fibonacci structures of the binding energies as a function of the impurity position in the superlattice and introduce a one-dimensional effective Coulomb potential that should be useful in the study of shallow-impurity states of Fibonacci superlattices, and other quasiperiodic semiconducting heterostructures, under the action of external electric and/or magnetic fields.57116573657

The Thermal Dissipation of LED Outdoor Lighting Luminaires: Comparative Analysis for a Real Case of Study

Today LED technology is being imposed, day by day, in our cities and homes as an efficient way of lighting. The performance of its lighting, durability, energy efficiency, and light, coupled with the economy of its use, is shifting to other classic forms of lighting. However, some problems associated with the durability of equipment associated with thermal dissipation and high-temperature problems, which end up affecting the light intensity and service life, are beginning to be detected. The objective of this paper is to compare the results obtained previously, at different contour temperatures, with the current practical results obtained with a FLUKETI25 thermal imaging camera. The theoretical results will be compared with the current results applied to the different luminaires. Where real thermal dissipation is studied, it is obtained for each of them in the laboratory of illumination with the thermographic camera FLUKE TI. The theoretical and experimental results are evaluated, and the results are discussed. This study shows that instead of LED technology, it is less risky for quality depreciation and durability of lighting if a project has already been achieved that favors optimal thermal dissipation, supported by the importance of choosing an appropriate design and appropriate materials

Impact and Learner Experience of a Technology Ecosystem as an Approach to Healthy Lifestyles: Erasmus+ SUGAPAS Project

The promotion of lifestyle habits in all ages is essential for the prevention of chronic non-communicable diseases. The aim of this study was to develop, validate, and disseminate an ecosystem that favors effective education on physical activity, a healthy lifestyle, and healthy nutritional habits. The sample consisted of 258 Spanish volunteer students. The evaluation of Ecosystem SUGAPAS was composed of two online surveys: MOOC and game surveys. The result of the MOOC survey reported a score above three points in all the sections (users’ opinion of the organization: 3.89 ± 0.71, 4.06 ± 0.64, and 4.01 ± 0.64; impact of the course on their day-to-day life: 3.62 ± 0.94; content present values: 3.96 ± 0.63, 3.69 ± 0.75, and 3.62 ± 0.94; usability of the platform: 3.75 ± 0.77, 3.96 ± 0.68, 4.06 ± 0.77, and 3.75 ± 0.84; relevance for professional development: 3.96 ± 0.63; overall opinion: 3.77 ± 0.57 points). The responses to all sections of the game evaluation reported a score between three and four points. A SUGAPAS ecosystem that includes new technologies for the promotion of a healthy lifestyle was developed, validated, and disseminated among the population. The users’ opinion, the impact of the ecosystem on their personal and professional development, and the usability of the platforms created make this ecosystem valid for its implementation for the proposed purposes

Bovine Neutrophils Release Extracellular Traps and Cooperate With Macrophages in Mycobacterium Avium Subsp. Paratuberculosis Clearance In Vitro

Mycobacterium avium subsp. paratuberculosis (Map) is the underlying pathogen causing bovine paratuberculosis (PTB), an enteric granulomatous disease that mainly affects ruminants and for which an effective treatment is needed. Macrophages are the primary target cells for Map, which survives and replicates intracellularly by inhibiting phagosome maturation. Neutrophils are present at disease sites during the early stages of the infection, but seem to be absent in the late stage, in contrast to healthy tissue. Although neutrophil activity has been reported to be impaired following Map infection, their role in PTB pathogenesis has not been fully defined. Neutrophils are capable of releasing extracellular traps consisting of extruded DNA and proteins that immobilize and kill microorganisms, but this mechanism has not been evaluated against Map. Our main objective was to study the interaction of neutrophils with macrophages during an in vitro mycobacterial infection. For this purpose, neutrophils and macrophages from the same animal were cultured alone or together in the presence of Map or Mycobacterium bovis Bacillus-Calmette-Guerin (BCG). Extracellular trap release, mycobacteria killing as well as IL-1 beta and IL-8 release were assessed. Neutrophils released extracellular traps against mycobacteria when cultured alone and in the presence of macrophages without direct cell contact, but resulted inhibited in direct contact. Macrophages were extremely efficient at killing BCG, but ineffective at killing Map. In contrast, neutrophils showed similar killing rates for both mycobacteria. Co-cultures infected with Map showed the expected killing effect of combining both cell types, whereas co-cultures infected with BCG showed a potentiated killing effect beyond the expected one, indicating a potential synergistic cooperation. In both cases, IL-1 beta and IL-8 levels were lower in co-cultures, suggestive of a reduced inflammatory reaction. These data indicate that cooperation of both cell types can be beneficial in terms of decreasing the inflammatory reaction while the effective elimination of Map can be compromised. These results suggest that neutrophils are effective at Map killing and can exert protective mechanisms against Map that seem to fail during PTB disease after the arrival of macrophages at the infection siteFunding was provided by Spanish central government and Basque research project PROBAK (RTA 2017-00089-00-00) and by the Departamento de Economia e Infraestructuras of the Basque Government. IL-A held a predoctoral grant from Departamento de Economia e Infraestructuras of the Basque Government (2017) and was granted an EMBO short-term fellowship (8407) and a FEMS research and training grant (FEMS-GO-2019-507). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publicatio

Internalization of the anti-carcinogenic IBB1, a major Bowman-Birk isoinhibitor from soybean (Glycine max), in HT29 colon cancer cells

Proceedings of the I Congress PIIISA celebrado en la Estación Experimental del Zaidín (Granada), en mayo de 2013.Protease inhibitors of the Bowman-Birk type, a major protease inhibitor family in legume seeds, which inhibit potently trypsin- and chymotrypsin-like proteases, are currently being investigated as colorectal chemopreventive agents. Although the therapeutic target/s and the action mechanism/s of Bowman-Birk inhibitors (BBI) have not yet been elucidated, the emerging evidence suggests that BBI exert their chemopreventive properties via protease inhibition; in this sense, serine proteases should be considered as primary targets in early stages of carcinogenesis. In this work, we have demonstrated that IBB1, a major protease inhibitor of the Bowman-Birk family in soybean (Glycine max), exerts anti-proliferative effect in human colorectal HT29 cancer cells at concentrations higher than 15 μM, in a dose dependent manner. By using confocal microscopy, we have demonstrated that IBB1 is taken up by HT29 colon cancer cells in a time-dependent manner, being the bulk of the internalized protease inhibitor localized in the cytoplasm where might interact with their potential therapeutic target/s.This work was supported by ERDF-co-financed grants AGL2011-26353 (Spanish Ministry of Economy and Competitiveness) and PE2010-CVI-5767 (Junta de Andalucía).Peer reviewe

Rendimiento y calidad de forraje del pasto ovillo (Dactylis glomerata L.) al variar la frecuencia e intensidad de pastoreo

The aim  of this  experiment was  to  study  the performance of Orchard  grass  (Dactylis glomerata L.)  based  on herbageyield  and  quality  under  different  grazing  management.  Three  frequencies  (2,  3  and  4  wk  in  spring  and  summer  and4,  5  and  6  wk  in  fall)  and  two  grazing  intensities  (hard:  3  to  5  cm  and  lax:  6  to  8  cm  of  residual  sward  height),  ina  randomized  block  design  with  three  replicates,  distributed  in  a  2  x  3  factorial  arrangement.  Cumulative  herbageyield  (CHY), net  herbage accumulation  rate  (NHAR), morphological  composition  and herbage  quality were  evaluated.The highest CHY and average NHAR were recorded with hard grazing every 4 and 6 wk. The highest seasonal herbageyield  (7,844  kg  DM  ha-1)  and  NHAR  (107  kg  DM  ha-1  d-1)  were  observed  during  summer  with  hard  grazing  every  4wk  (P0.05).Across  all  seasons,  hard  grazing  gave  higher  forage  yield  than  lax  grazing,  being  different  in  spring  and  summer(P<0.05). In overall, total protein and in vitro dry matter digestibility tended to decrease as grazing interval increased,being  on  average  20  and  65  %,  respectively. It  was  concluded  that  to  obtain  the  highest  herbage yield  and  quality,orchard  grass  must  be  grazed  hard  every  4  wk  in  spring-summer  and  every  5  wk  in  fall.El objetivo fue determinar el rendimiento y calidad del pasto ovillo (Dactylis glomerata L.), al variar la frecuencia e intensidad de pastoreo. Se evaluaron tres frecuencias (2, 3 y 4 semanas en primavera y verano y 4, 5 y 6 semanas durante otoño) y dos intensidades de pastoreo (severa: 3 a 5 cm y ligera: 6 a 8 cm de altura de forraje residual). Se evaluó el rendimiento de forraje, tasa de acumulación neta de forraje (TANF), composición morfológica y calidad del forraje. El mayor rendimiento acumulado y TANF se presentaron con pastoreo severo cada 4 y 6 semanas y en ambas intensidades cada 3 y 5 semanas. El mayor rendimiento estacional (7,844; 7,699; 7114 kg MS ha-1) se presentó en verano en las mismas frecuencias e intensidades de pastoreo, respectivamente (P<0.05). Durante primavera no se observaron diferencias en rendimiento y TANF entre frecuencias de pastoreo. En otoño la frecuencia de 5 semanas superó a la de 4 (P<0.05), pero no diferente a la de 6 semanas. En todas las estaciones del año la intensidad de pastoreo severo superó a la ligera, siendo diferentes en primavera y verano (P<0.05). En general, el contenido de proteína total y digestibilidad in vitro de la materia seca tendieron a disminuir conforme aumentó el intervalo de pastoreo, siendo en promedio de 20 y 65 %, respectivamente. Se concluye que para obtener el mayor rendimiento y calidad de forraje, los pastoreos deben realizarse cada 4 semanas durante primavera-verano y cada 5 semanas en otoño, a una intensidad severa