| 1. |
- Alvarez-Rodríguez, Manuel, et al.
(författare)
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The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa.
- 2013
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Ingår i: Reproduction, Fertility and Development. - : CSIRO Publishing. - 1031-3613 .- 1448-5990. ; 25:8, s. 1185-1193
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Tidskriftsartikel (refereegranskat)abstract
- Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.
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| 2. |
- Alvarez-Rodriguez, Manuel, et al.
(författare)
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Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition.
- 2011
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 75:8, s. 1561-1565
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Tidskriftsartikel (refereegranskat)abstract
- We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.
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| 3. |
- Alvarez-Rodríguez, Manuel, et al.
(författare)
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The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.
- 2013
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 79:3, s. 541-550
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Tidskriftsartikel (refereegranskat)abstract
- The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).
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| 4. |
- Anel-López, Luis, et al.
(författare)
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Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa
- 2012
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Ingår i: Animal Reproduction Science. - : Elsevier. - 0378-4320 .- 1873-2232. ; 135:1-4, s. 37-46
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Tidskriftsartikel (refereegranskat)abstract
- The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39°C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.
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| 5. |
- de Paz, Paulino, et al.
(författare)
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Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).
- 2012
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Ingår i: Reproduction in domestic animals. - : Blackwell Verlag. - 0936-6768 .- 1439-0531. ; 47:1, s. 105-112
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Tidskriftsartikel (refereegranskat)abstract
- In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.
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| 6. |
- Franco-Gonzalez, Juan Felipe, et al.
(författare)
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Conformational flexibility of the ErbB2 ectodomain and trastuzumab antibody complex as revealed by molecular dynamics and principal component analysis.
- 2013
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Ingår i: Journal of Molecular Modeling. - : Springer Science and Business Media LLC. - 1610-2940 .- 0948-5023. ; 19:3, s. 1227-1236
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor 2 (ErbB2) is a transmembrane oncoprotein that is over expressed in breast cancer. A successful therapeutic treatment is a monoclonal antibody called trastuzumab which interacts with the ErbB2 extracellular domain (ErbB2-ECD). A better understanding of the detailed structure of the receptor-antibody interaction is indeed of prime interest for the design of more effective anticancer therapies. In order to discuss the flexibility of the complex ErbB2-ECD/trastuzumab, we present, in this study, a multi-nanosecond molecular dynamics simulation (MD) together with an analysis of fluctuations, through a principal component analysis (PCA) of this system. Previous to this step and in order to validate the simulations, we have performed a detailed analysis of the variable antibody domain interactions with the extracellular domain IV of ErbB2. This structure has been statically elucidated by x-ray studies. Indeed, the simulation results are in excellent agreement with the available experimental information during the full trajectory. The PCA shows eigenvector fluctuations resulting in a hinge motion in which domain II and C(H) domains approach each other. This move is likely stabilized by the formation of H-bonds and salt bridge interactions between residues of the dimerization arm in the domain II and trastuzumab residues located in the C(H) domain. Finally, we discuss the flexibility of the MD/PCA model in relation with the static x-ray structure. A movement of the antibody toward the dimerization domain of the ErbB2 receptor is reported for the first time. This finding could have important consequences on the biological action of the monoclonal antibody.
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| 7. |
- Franco-Gonzalez, Juan Felipe, et al.
(författare)
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Exploring the dynamics and interaction of a full ErbB2 receptor and Trastuzumab-Fab antibody in a lipid bilayer model using Martini coarse-grained force field.
- 2014
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Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; 28:11, s. 1093-1107
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Tidskriftsartikel (refereegranskat)abstract
- Coarse grained (CG) modeling has been applied to study the influence of the Trastuzumab monoclonal antibody on the structure and dynamics of the full ErbB2 receptor dimer, including the lipid bilayer. The usage of CG models to study such complexes is almost mandatory, at present, due to the large size of the whole system. We will show that the Martini model performs satisfactorily well, giving results well-matched with those obtained by atomistic models as well as with the experimental information existing on homolog receptors. For example, the extra and intracellular domains approach the bilayer surface in both the monomer and dimer cases. The Trastuzumab-Fab hinders the interaction of the receptors with the lipid bilayer. Another interesting effect of the antibody is the disruption of the antiparallel arrangement of the juxtamembrane segments in the dimer case. These findings might help to understand the effect of the antibody on the receptor bioactivity.
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| 8. |
- Franco-Gonzalez, Juan Felipe, et al.
(författare)
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Simulation of homology models for the extracellular domains (ECD) of ErbB3, ErbB4 and the ErbB2-ErbB3 complex in their active conformations.
- 2013
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Ingår i: Journal of Molecular Modeling. - : Springer Science and Business Media LLC. - 1610-2940 .- 0948-5023. ; 19:2, s. 931-941
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Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor receptors (EGFR) are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this work, we provide models based on homology for the extracellular domains (ECD) of ErbB3 and ErbB4 in their active conformations, including a Heregulin ligand, followed by further refinement of the models by molecular dynamics simulations at atomistic scale. We compare the results with a model built for ErbB2 based on crystallographic information and analyze the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of domain IV. Finally, we refine a model for the interaction of the ECDs corresponding to a ErbB2-ErbB3 heterodimer, which is widely recognized to have a high impact in cancer development.
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| 9. |
- Gil-Castell, O., et al.
(författare)
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Hydrothermal ageing of polylactide/sisal biocomposites. Studies of water absorption behaviour and Physico-Chemical performance
- 2014
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Ingår i: Polymer degradation and stability. - : Elsevier BV. - 0141-3910 .- 1873-2321. ; 108, s. 212-222
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Tidskriftsartikel (refereegranskat)abstract
- An accelerated hydrothermal degrading test was designed in order to analyse the synergic effect of water and temperature on PLA/sisal biocomposites with and without coupling agent. As well, the physicochemical properties of biocomposites were monitored along the hydrothermal test by means of Scanning Electron Microscopy, Size Exclusion Chromatography and Differential Scanning Calorimetry. The addition of fibre induced higher water absorption capability and promoted physical degradation, as observed in the surface topography. During the processing of biocomposites and throughout the hydrothermal ageing, a reduction of molecular weight due to chain scission was found. As a consequence, a faster formation of crystalline domains in the PIA matrix occurred the higher the amount of fibre was, which acted as a nucleating agent. Higher crystallinity was considered as a barrier against the advance of penetrant and a reduction in the diffusion coefficient was shown. The addition of coupling agent presented a different influence depending on the composition, showing an inflection point around 20% of sisal fibre.
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| 10. |
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