Ex vivo exposure to titanium dioxide and silver nanoparticles mildly affect sperm of gilthead seabream (Sparus aurata) - A multiparameter spermiotoxicity approach.

Nanoparticles (NP) are potentially reprotoxic, which may compromise the success of populations. However, the reprotoxicity of NP is still scarcely addressed in marine fish. Therefore, we evaluated the impacts of environmentally relevant and supra environmental concentrations of titanium dioxide (TiO2: 10 to 10,000 µg·L-1) and silver NP (Ag: 0.25 to 250 µg·L-1) on the sperm of gilthead seabream (Sparus aurata). We performed short-term direct exposures (ex vivo) and evaluated sperm motility, head morphometry, mitochondrial function, antioxidant responses and DNA integrity. No alteration in sperm motility (except for supra environmental Ag NP concentration), head morphometry, mitochondrial function, and DNA integrity occurred. However, depletion of all antioxidants occurred after exposure to TiO2 NP, whereas SOD decreased after exposure to Ag NP (lowest and intermediate concentration). Considering our results, the decrease in antioxidants did not indicate vulnerability towards oxidative stress. TiO2 NP and Ag NP induced low spermiotoxicity, without proven relevant ecological impacts.

Glycine rich segments adopt polyproline II helices: Implications for biomolecular condensate formation.

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Based on NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772).

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p < 0.001), however, methanol had the lowest protective capacity while DMSO the highest. Afterwards, two different thawing rates (1 min at 5 °C; and 25 s at 10 °C) were tested using only 10% DMSO and 10% propanediol. Both for the DMSO and propanediol, there were no significant differences (p > 0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2-4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p < 0.001). At a concentration of 5 × 105 spermatozoa:egg, similar fertilizations rates to the fresh sperm were obtained. The presence of the cryoprotectant DMSO during the 4 h contact time did not affect the fertilization rate or the percentage of embryos with abnormal cleavage (p > 0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (-14.05 °C/min) and 7.5 cm (-5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.

Chiral amplification of disodium cromoglycate chromonics induced by a codeine derivative.

Chromonic liquid crystals (CLC) are lyotropic phases formed by discotic mesogens in water. Simple chiral dopants such as amino acids have been reported to turn chromonic liquid crystals into their cholesteric counterparts. Here we report a chirality amplification effect in the nematic phase of a 9 wt% disodium cromoglycate (DSCG) lyotropic liquid crystal (LLC) upon doping with a water-soluble codeine derivative. The transition on cooling the isotropic to the nematic phase showed the presence of homochiral spindle-shaped droplets (tactoids). NMR DOSY experiments on a triple gradient probe revealed a small degree of diffusion anisotropy for the alkaloid embedded in the liquid crystal structure. These results in combination with XRD, CD and POM experiments agree with a supramolecular aggregation model based on simple columnar stacks.

Busulfan administration produces sublethal effects on somatic tissues and inhibits gametogenesis in Senegalese sole juveniles.

Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20÷-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg⁻¹ busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.

Effect of two sulfur-containing amino acids, taurine and hypotaurine in European sea bass (Dicentrarchus labrax) sperm cryopreservation.

In the present work, taurine and hypotaurine were evaluated as potential additives to improve European sea bass (Dicentrarchus labrax) sperm quality after cryopreservation. For cryopreservation, three different extenders were used: control extender (NAM), supplemented with 1mM taurine or supplemented with 1mM hypotaurine, all of them containing 10% Me2SO as cryoprotectant. To evaluate sperm quality of fresh and thawed sperm, motility (CASA: computer assisted sperm analysis), viability (SYBR Green/propidium iodide), lipid peroxidation (malondialdehyde level), protein oxidation (carbonyl content), glutathione peroxidase, glutathione reductase and superoxide dismutase activities and DNA fragmentation (comet assay) were quantified. The result demonstrated that 1 mM hypotaurine supplemented extender increased total motility (30.1 ± 3.2%), and that 1 mM taurine extender produced higher velocity (18.1 ± 2.6 µm/s) and linearity (46.0 ± 4.8%) than the control extender (21.8 ± 3.2%, 15.5 ± 1.3 µm/s, 41.8 ± 2.4%, respectively). Cell viability, lipid peroxidation and protein oxidation were not statistically different between treatments. Similar results were obtained for glutathione peroxidase and superoxide dismutase activities. Only glutathione reductase showed differential activity before and after freezing, increasing its activity in thawed sperm. Regarding the comet assay results, taurine and hypotaurine significantly reduced DNA fragmentation (52.8 ± 0.9% and 51.8 ± 0.9%, respectively) in comparison to the control (55.7 ± 0.8%). In conclusion, for European sea bass sperm cryopreservation, extenders supplemented with 1 mM taurine and 1 mM hypotaurine improved some parameters of sperm quality after thawing, resulting in better motility and lower DNA damage than the control, two very important factors related to fertilization success.

Sea bass sperm freezability is influenced by motility variables and membrane lipid composition but not by membrane integrity and lipid peroxidation.

Cryopreserved sperm quality depends on the characteristics of fresh sperm. Thus, it is necessary to establish a group of variables to predict the cryopreservation potential of the fresh samples with the aim of optimizing resources. Motility, viability, lipid peroxidation and lipid profile of European sea bass (Dicentrarchus labrax) sperm were determined before and after cryopreservation to establish which variables more accurately predict the sperm cryopreservation potential in this species. Cryopreservation compromised sperm quality, expressed as a reduction of motility (46.5 ± 2.0% to 35.3 ± 2.5%; P<0.01) and viability (91.3 ± 0.7% to 69.9 ± 1.6%; P<0.01), and produced an increase in lipid peroxidation (2.4 ± 0.4 to 4.0 ± 0.4 µmoles MDA/mill spz; P<0.01). Also, significant changes were observed in the lipid composition before and after freezing, resulting in a reduction in the cholesterol/phospholipids ratio (1.4 ± 0.1 to 1.1 ± 0.0; P<0.01), phosphatidylcholine (47.7 ± 0.8% to 44.2 ± 0.8%; P<0.01) and oleic acid (8.7 ± 0.2% to 8.3 ± 0.2%; P<0.05) in cryopreserved sperm, as well as an increase in lysophosphatidylcholine (4.4 ± 0.3% to 4.8 ± 0.3%; P<0.01) and C24:1n9 fatty acid (0.5 ± 0.1% to 0.6 ± 0.1%; P<0.05). Motility, velocity, cholesterol/phospholipids ratio, monounsaturated fatty acids and the n3/n6 ratio were positively correlated (P<0.05) before and after freezing, whereas, viability and lipid peroxidation were not correlated. Motility and the cholesterol/phospholipids (CHO/PL) ratio were negatively correlated (P<0.05) with each other and the CHO/PL ratio was positively correlated (P<0.05) with lipid peroxidation. Therefore, the results demonstrated that motility and plasma membrane lipid composition (CHO/PL) were the most desirable variables determined in fresh samples to predict cryo-resistance in European sea bass sperm, taking into account the effect of both on cryopreserved sperm quality.

Understanding the ion jelly conductivity mechanism.

The properties of the light flexible device, ion jelly, which combines gelatin with an ionic liquid (IL) were recently reported being promising to develop safe and highly conductive electrolytes. This article aims for the understanding of the ion jelly conductive mechanism using dielectric relaxation spectroscopy (DRS) in the frequency range 10(-1)-10(6) Hz; the study was complemented with differential scanning calorimetry (DSC) and pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. The room temperature ionic liquid 1-butyl-3-methylimmidazolium dicyanamide (BMIMDCA) used as received (1.9% w/w water content) and with 6.6% (w/w) of water content and two ion jellies with two different ratios BMIMDCA/gelatin/water % (w/w), IJ1 (41.1/46.7/12.2) and IJ3 (67.8/25.6/6.6), have been characterized. A glass transition was detected by DSC for all materials allowing for classifying them as glass formers. For the ionic liquid, it was observed that the glass transition temperature decreases with the increase of water content. While in subsequent calorimetric runs crystallization was observed for BMIMDCA with negligible water content, no crystallization was detected for any of the ion jelly materials upon themal cycling. To the dielectric spectra of all tested materials, both dipolar relaxation and conductivity contribute; at the lowest frequencies, electrode and interfacial polarization highly dominate. Conductivity, which manifests much more intensity relative to dipolar reorientations, strongly evidences subdiffusive ion dynamics at high frequencies. From dielectric measures, transport properties as mobility and diffusion coefficients were extracted. Data treatment was carried out in order to deconvolute the average diffusion coefficients estimated from dielectric data in its individual contributions of cations (D(+)) and anions (D(-)). The D(+) values thus obtained for IJ3, the ion jelly with the highest IL/gelatin ratio, cover a large temperature range up to room temperature and revealed excellent agreement with direct measurements from PFG NMR, obeying to the same VFT equation. For BMIMDCA(6.6%water), which has the same water amount as IJ3, the diffusion coefficients were only estimated from DRS measurements over a limited temperature range; however, a single VFT equation describes both DRS and PFG NMR data. Moreover, it was found that the diffusion coefficients and mobility are similar for the ionic liquid and IJ3, which points to a role of both water and gelatin weakening the contact ion pair, facilitating the translational motion of ions and promoting its dissociation; nevertheless, it is conceivable the existence of a critical composition of gelatin that leads to those properties. The VFT temperature dependence observed for the conductivity was found to be determined by a similar dependence of the mobility. Both conductivity and segmental motion revealed to be correlated as inferred by the relatively low values of the decoupling indexes. The obtained results show that ion jelly could be in fact a very promising material to design novel electrolytes for different electrochemical devices, having a performance close to the IL but presenting an additional stability regarding electrical measurements and resistance against crystallization relative to the bulk ionic liquid.

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm.

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 µm/sec, 18.3 ± 1.7 µm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 µm/sec, 19.5 ± 1.6 µm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 µm/sec, 13.8 ± 1.7 µm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.

Changes in Solea senegalensis sperm quality throughout the year.

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March-May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.

The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation.

During cryopreservation, dilution in the extender media reduces the seminal plasma constituents being cells more vulnerable to oxidative stress. Vitamins (C and E) and the amino acids taurine and hypotaurine are powerful antioxidants naturally present in seminal plasma. Whether their effect may improve sperm quality and reduce sperm DNA damage after cryopreservation in fish sperm still remains unclear. Thus, the aim of the present work was to analyse the effect of extender supplementation with several antioxidant components on post-thawed sperm motility, viability and DNA integrity of two commercial species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Sperm collected from ten to twelve individuals was cryopreserved in ten different extenders containing: taurine and hypotaurine (1 and 10mM), ascorbic acid (1 and 10mM), α-tocoferol (0.1 and 0.5 mM) or 1 ml/l of a commercial cell antioxidant supplement. Cell viability, motility and DNA fragmentation were determined in post-thawed samples. Addition of antioxidants (vitamins and amino acids) to D. labrax and S. aurata extenders did not significantly increase the parameters of motility (TM, PM, VCL, VSL and Lin) or viability, although 1mM taurine slightly increased the percentage of motile cells (TM) in S. aurata. DNA fragmentation (DNA in tail and Olive tail moment) in D. labrax sperm was higher in treatments containing vitamins than amino acids or control. However in S. aurata sperm, antioxidants especially taurine and hypotaurine, significantly reduced both DNA fragmentation parameters, protecting DNA against strand breaks. These results suggest a species-specific effect depending on the type of antioxidants used.

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity.

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20ß-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 µg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.

Effect of cryopreservation on fish sperm subpopulations.

The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me(2)SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5ml straws, and 1.8ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8cm above the liquid nitrogen surface for the straws and 1, 2 and 4cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 - slow non-linear spermatozoa, SP2 - slow linear spermatozoa and SP3 - fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15s after activation and was also the one showing a greater decrease in time, being the least represented after 60s. According to the applied univariate general linear model, samples frozen in straws with 5% Me(2)SO and in cryovials with 10% Me(2)SO at 2 and 1cm from the LN(2,) respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods.

The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

Asymmetric intramolecular aldol reactions of substituted 1,7-dicarbonylic compounds. A mechanistic study.

The diastereo- and enantioselectivity obtained experimentally by List on the proline-catalyzed intramolecular aldol reaction of substituted 1,7-dicarbonylic compounds was accurately predicted using density functional theory methods at the B3LYP/6-31++G** level. A polarizable continuum model was used to describe solvent effects. The theoretical data agree in good extension with List's experimental results, both in enantioselectivity and diastereoselectivity, and allow for the confirmation of our previous rationalization of the main factors contributing to the reaction selectivity. While the enantioselectivity results from an important electrostatic contact between the forming alkoxyde group and the proline moiety, the calculated diasteroselectivity results from several steric contacts that can be established between the different substituents and from their relative orientation in respect to the ring conformation. However, for dialdehydes that can originate two diastereomeric enamine intermediates, the proline attack and the immonium formation steps can also be of major importance in the rationalization of the final reaction selectivity, as is the case in two of the six studied systems. The obtained data allows for a full rationalization of the known experimental systems as well as for the extrapolation to new ones with variable substitution at the carbonylic chain.

Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

Development of PMMA membranes functionalized with hydroxypropyl-beta-cyclodextrins for controlled drug delivery using a supercritical CO(2)-assisted technology.

Cyclodextrin-containing polymers have proved themselves to be useful for controlled release. Herein we describe the preparation of membranes of poly(methylmethacrylate) (PMMA) containing hydroxypropyl-beta-cyclodextrins (HP-beta-CDs) using a supercritical CO(2)-assisted phase inversion method, for potential application as drug delivery devices. Results are reported on the membrane preparation, physical properties, and drug elution profile of a model drug. The polymeric membranes were obtained with HP-beta-CD contents ranging from 0 to 33.4 wt%, by changing the composition of the casting solution, and were further impregnated with ibuprofen using supercritical carbon dioxide (scCO(2)) in batch mode. The influence of the membrane functionalization in the controlled release of ibuprofen was studied by performing in vitro experiments in buffer solution pH at 7.4. The release of the anti-inflammatory drug could be tuned by varying the cyclodextrin content on the membranes.