Impact of TGF-ß family-related growth factors on chondrogenic differentiation of adipose-derived stem cells isolated from lipoaspirates and infrapatellar fat pads of osteoarthritic patients.

The success of cell-based approaches for the treatment of cartilage defects requires an optimal autologous cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. The objective of this study was to compare the chondrogenic capacity of mesenchymal stem cells (MSCs) isolated from lipoaspirates (ASCs) and the infrapatellar fat pad (IFPSCs) of osteoarthritic patients and treated with transforming growth factor (TGF)-ß family-related growth factors. Cells were cultured for 6 weeks in a 3D pellet culture system with the chimeric activin A/bone morphogenic protein (BMP)-2 ligand (AB235), the chimeric nodal/BMP-2 ligand (NB260) or BMP-2. To investigate the stability of the new cartilage, ASCs-treated pellets were transplanted subcutaneously into severe combined immunodeficiency (SCID) mice. Histological and immunohistochemical assessment confirmed that the growth factors induced cartilage differentiation in both isolated cell types. However, reverse transcription-quantitative PCR results showed that ASCs presented a higher chondrogenic potential than IFPSCs. In vivo results revealed that AB235-treated ASCs pellets were larger in size and could form stable cartilage-like tissue as compared to NB260-treated pellets, while BMP-2-treated pellets underwent calcification. The chondrogenic induction of ASCs by AB235 treatment was mediated by SMAD2/3 activation, as proved by immunofluorescence analysis. The results of this study indicated that the combination of ASCs and AB235 might lead to a cell-based cartilage regeneration treatment.

Temperature and rainfall are related to fertility rate after spring artificial insemination in small ruminants.

A total number of 1092 artificial inseminations (AIs) performed from March to May were documented over four consecutive years on 10 Payoya goat farms (36° N) and 19,392 AIs on 102 Rasa Aragonesa sheep farms (41° N) over 10 years. Mean, maximum, and minimum ambient temperatures, mean relative humidity, mean solar radiation, and total rainfall on each insemination day were recorded. Overall, fertility rates were 58 % in goats and 45 % in sheep. The fertility rates of the highest and lowest deciles of each of the meteorological variables indicated that temperature and rainfall had a significant effect on fertility in goats. Specifically, inseminations that were performed when mean (68 %), maximum (68 %), and minimum (66 %) temperatures were in the highest decile, and rainfall was in the lowest decile (59 %), had a significantly (P < 0.0001) higher proportion of does that became pregnant than did the ewes in the lowest decile (56, 54, 58, and 49 %, respectively). In sheep, the fertility rates of the highest decile of mean (62 %), maximum (62 %), and minimum (52 %) temperature, RH (52 %), THI (53 %), and rainfall (45 %) were significantly higher (P < 0.0001) than were the fertility rates among ewes in the lowest decile (46, 45, 45, 45, 46, and 43 %, respectively). In conclusion, weather was related to fertility in small ruminants after AI in spring. It remains to be determined whether scheduling the dates of insemination based on forecasted temperatures can improve the success of AI in goats and sheep.

Chondrocytes extract from patients with osteoarthritis induces chondrogenesis in infrapatellar fat pad-derived stem cells.

OBJECTIVE: Infrapatellar fat pad of patients with osteoarthritis (OA) contains multipotent and highly clonogenic adipose-derived stem cells that can be isolated by low invasive methods. Moreover, nuclear and cytoplasmic cellular extracts have been showed to be effective in induction of cell differentiation and reprogramming. The aim of this study was to induce chondrogenic differentiation of autologous mesenchymal stem cells (MSCs) obtained from infrapatellar fat pad (IFPSCs) of patients with OA using cellular extracts-based transdifferentiation method. DESIGN: IFPSCs and chondrocytes were isolated and characterized by flow cytometry. IFPSCs were permeabilized with Streptolysin O and then exposed to a cell extract obtained from chondrocytes. Then, IFPSCs were cultured for 2 weeks and chondrogenesis was evaluated by morphologic and ultrastructural observations, immunologic detection, gene expression analysis and growth on 3-D poly (dl-lactic-co-glycolic acid) (PLGA) scaffolds. RESULTS: After isolation, both chondrocytes and IFPSCs displayed similar expression of MSCs surface makers. Collagen II was highly expressed in chondrocytes and showed a basal expression in IFPSCs. Cells exposed to chondrocyte extracts acquired a characteristic morphological and ultrastructural chondrocyte phenotype that was confirmed by the increased proteoglycan formation and enhanced collagen II immunostaining. Moreover, chondrocyte extracts induced an increase in mRNA expression of chondrogenic genes such as Sox9, L-Sox5, Sox6 and Col2a1. Interestingly, chondrocytes, IFPSCs and transdifferentiated IFPSCs were able to grow, expand and produce extracellular matrix (ECM) on 3D PLGA scaffolds. CONCLUSIONS: We demonstrate for the first time that extracts obtained from chondrocytes of osteoarthritic knees promote chondrogenic differentiation of autologous IFPSCs. Moreover, combination of transdifferentiated IFPSCs with biodegradable PLGA 3D scaffolds can serve as an efficient system for the maintenance and maturation of cartilage tissue. These findings suggest its usefulness to repair articular surface in OA.

Genetic variation of adaptation to heat stress in two Spanish dairy goat breeds.

Monthly test-day records of milk yield and composition in Murciano-Granadina (MG) and Payoya (PYA) dairy goats were combined with weather data from meteorological stations, to analyse the effects of heat stress on dairy traits, measured with an index of temperature and relative humidity (THI). A 'repeatability animal model' and a 'reaction norm animal model' were used to estimate genetic (co)variance components. Estimates obtained with both models were very similar. The h(2) of daily yields in MG did not vary throughout the THI scale, but the pattern of variation of content traits showed negative trends for increasing THI values. In PYA, a slight positive tendency throughout the THI scale was observed for the same traits. The genetic correlations between subsequent points in the THI scale were lower than 0.80 when they were computed between low and high THI points. The same reaction norm was observed for all traits. Using the 'reaction norm animal model', it was possible to identify those animals that show the same performance throughout the THI trajectory (robust) and those with varying performances (tolerant and non-tolerant to heat stress). Results in this study also show that heat tolerance decreases, while the genetic level for milk traits increases. Losses because of heat stress were equivalent to 1.9 and 3.1% in the yearly yield of fat and protein for MG and PYA, respectively.

Co-formulants in plant protection products: An analytical approach to their determination by gas chromatography-high resolution mass accuracy spectrometry.

In the present study, 12 volatile benzene and naphthalene derived co-formulants were identified by suspect screening and unknown analysis in 14 plant protection products (PPPs) corresponding to several types of formulations, as emulsifiable concentrates (EC), suspension concentrates (SC), dispersible concentrates (DC) and ZC, which is a mixture of a capsule suspension (CS) in an SC, containing either difenoconazole or chlorantraniliprole as main active ingredients. The selected technique was gas chromatography coupled to Q-Orbitrap high resolution mass accuracy spectrometry (GC-Q-Orbitrap-MS), providing efficient separation and detection of all identified compounds. Finally, 42 compounds were tentatively identified, and 12 of them were confirmed and quantified using analytical standards. Results showed that the applied methodology was able to detect these co-formulants at concentrations as low as 0.03 g/L (tert-butylbenzene), encompassing a wide concentration range, up to 9.63 g/L (pentamethylbenzene). Pentamethylbenzene was the only compound detected in all studied samples.

Random regression analysis of milk yield and milk composition in the first and second lactations of Murciano-Granadina goats.

Records from the milk recording scheme of Spanish Murciano-Granadina goats were studied to estimate genetic (co)variance components and breeding values throughout the first and second lactations. The data used consisted of 49,696 monthly test-day records of milk (MY), protein (PY), fat (FY), and dry matter (DMY) yields from 5,163 goats, distributed in 20 herds, offspring of 2,086 does and 206 bucks. These records were analyzed by 2-trait random regression models (RRM) and a repeatability test-day model (RTDM). At the middle of lactation, heritability estimates for MY, DMY, and FY obtained with RTDM were larger than those estimated with RRM, and the opposite was true for PY. The RRM estimates of heritability for MY, FY, and PY were very similar throughout the trajectories of both lactations. Heritability estimates for DMY decreased through the lactation period. The genetic correlations between the first and second lactation records estimated for all traits by RRM were positive and ranged from 0.43 to 0.80 throughout the lactation curves. The correlation between BV estimated with RTDM and RRM was 0.742 for MY and 0.664 for DMY. The RRM could be a useful alternative to RTDM for the prediction of BV in this breed.

Solid phase microextraction and gas chromatography coupled to magnetic sector high resolution mass spectrometry for the ultra-trace determination of contaminants in surface water.

With the aim of monitoring water quality according to the regulations established by the European Union it would be necessary to implement analytical methodologies capable of simultaneously determining a broad range of organic pollutants at ultra-trace levels, allowing for increased sample throughput. In addition, the high number of samples to be analyzed requires a particular focus on setting up fully automated analytical methodologies. In view of that, this study is aimed at the development of a complete automated procedure for the ultra-trace determination of certain pesticides, polycyclic aromatic hydrocarbons (PAHs), brominated diphenyl ethers (BDEs) and polychlorinated biphenyls (PCBs) in surface waters. The proposed method is based on an on-line combination of solid phase microextraction (SPME) and gas chromatography coupled to double-focusing magnetic sector high resolution mass spectrometry (GC-HRMS). SPME as well as GC-HRMS conditions were optimized to achieve maximum extraction efficiency and sensitivity, which was reinforced by using multiple ion detection (MID) as acquisition mode. Using only 19mL of water and with minimum sample manipulation, the method allowed for the determination of 53 compounds exhibiting good linearity (R2>0.99), recoveries between 84 and 118% and relative standard deviation (RSD) values <20% for intra-day and inter-day precision. In addition, the method provides quantification limits (LOQs) between 0.1-50ngL-1, lower than the Environmental Quality Standards (EQS) fixed by Directive 2013/39/EC. Finally, the method was successfully applied to determine target contaminants in Almería surface water compartments, detecting dioxin-like PCBs, BDEs and some pesticides.

Storage temperature and sucrose concentrations affect ram sperm quality after vitrification.

The aim of this study was to analyse the characteristics of ram spermatozoa subjected to varying concentrations of sucrose, and the influence of storage temperature (22°C or 5°C) prior to vitrification. Ejaculated semen was diluted in TCFEY (tris-citric acid-fructose 20% egg yolk), and two aliquots were prepared at a final concentration of 100×106spz/ml, one maintained at room temperature (22°C) and the other at 5°C. In the first experiment, the toxicity of sucrose diluents on the sperm was analysed; sperm samples at different temperatures were diluted (1:2) in TCF-BSA 2% (control) or in the same extender supplemented with various sucrose concentrations (0.4M, 0.6M and 0.8M). The effects of vitrification were studied in the second experiment, where sperm samples were mixed with different concentrations of cryoprotectants (sucrose) and vitrified by being plunged directly into liquid nitrogen. In both experiments, the sperm quality was assessed by measuring motility, morphology, membrane functionality (HOST), viability, acrosome integrity and DNA fragmentation. The toxicity test revealed significant differences (p≤0.05) when different sucrose concentrations were used; lower total and progressive motility, normal morphology and membrane functionality were noted when sucrose concentration was higher, compared to the control treatment. Samples maintained at room temperature showed significantly (p≤0.05) higher viability than samples stored at 5°C. In contrast, although the quality of vitrified sperm was drastically decreased in comparison with fresh sperm, sucrose was associated with greater total motility, viability and membrane functionality. This improvement was closely linked to the temperature at which the sperm had been previously maintained, showing higher values when sperm was stored at 5°C. The main conclusions to be drawn from the study are therefore that sucrose shows promising potential as a cryoprotectant, and storing samples at 5°C is linked to improved sperm quality following vitrification.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Application of gas chromatography-tandem mass spectrometry to the analysis of pesticides in fruits and vegetables.

A new analytical method was devised using gas chromatography with tandem mass spectrometry (GC-MS-MS) for the routine analysis of 31 multi-class pesticide residues and approximately 8000 fresh fruit and vegetable samples (green bean, cucumber, pepper, tomato, eggplant, watermelon, melon, and marrow). Extraction of the pesticides with dichloromethane was carried out. The optimal ionization mode, either electron impact or chemical ionization, was selected for each pesticide in the same run. Carbofrit was used in the liner and combined with the selectivity of the detector this avoided additional clean-up. Thus, not only was money and time saved, the uncertainty of the method was decreased in its application to routine analysis. The average recoveries in cucumber obtained for each pesticide ranged between 71 and 119% at two different fortification levels (n=10 each) that ranged between 7 and 300 ng g(-1) (depending on the pesticide). The relative standard deviation was lower than 19% for all compounds tested. The calculated limits of detection and quantification were typically <1 ng g(-1) which were much lower than the maximum residue levels established by European legislations.

Pesticide trace analysis using solid-phase extraction and gas chromatography with electron-capture and tandem mass spectrometric detection in water samples.

Gas chromatography (GC) with electron-capture detection (ECD), mass spectrometry (MS) and tandem mass spectrometry (MS-MS) were employed for the identification of 12 pesticides in water samples. For this purpose, a solid-phase extraction procedure with C18 cartridges was used, optimising the breakthrough volume and the saturation concentration. In GC-MS-MS, the lowest detectable concentrations for the pesticides were between 2 and 26 ng l(-1), recoveries ranged from 70 to 133% in water samples spiked at 100 ng l(-1) and the relative standard deviations were in the range 5.3 to 17.4%. The proposed analytical methodology was applied to analyse pesticides in wetland samples from Almería (Spain).

Reduction of analysis time in gas chromatography. Application of low-pressure gas chromatography-tandem mass spectrometry to the determination of pesticide residues in vegetables.

An alternative to conventional capillary gas chromatography (GC) is evaluated as a new approach to determine pesticide residues in vegetables. Low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS-MS) is proposed after a fast and simple extraction of the vegetable samples with dichloromethane and without clean up. The use of the above-mentioned GC technique reduced the total time required to determine 72 pesticides to less than half the present time (31 min), increasing the capability of a monitoring routine laboratory. The use of guard column and plug of carbofrit into the glass liner in combination with LP-GC was evaluated. The method was validated with limits of quantitation low enough to determine the pesticide residues at concentrations below the maximum residue levels stated by legislation. In order to assess its applicability to the analysis of real samples, 25 vegetable samples previously determined using conventional-capillary GC-MS-MS were analysed by LP-GC-MS-MS. The results obtained with the compared techniques showed differences lower than 0.01 mg kg(-1).

Gas chromatographic-tandem mass spectrometric analytical method for the study of inhalation, potential dermal and actual exposure of agricultural workers to the pesticide malathion.

New analytical gas chromatographic-tandem mass spectrometric approaches have been developed for assessing both potential and actual exposure of agricultural workers to malathion. The metabolites alpha- and beta-malathion monocarboxylic acids have been determined after a derivatisation process in order to obtain their hexafluoroisopropyl esters. Whole body dosimetry was used for potential dermal exposure assessment. Potential exposure by inhalation was estimated using personal air samplers and polyurethane foam plugs as sorbents. The intern dose measurements were carried out by analysing samples of urine after solid-phase extraction with C18. The recoveries of the analytes of the three matrices were between 90 and 102%. Quantification limits were lower than 0.24 ng L(-1). The proposed methods have been applied to evaluate potential and actual exposure of applicators spraying malathion in greenhouses.

Excretion study of endosulfan in urine of a pest control operator.

A study of endosulfan excretion in human urine was carried out by using an agricultural worker involved with spraying, wearing protective overall, gloves and breathing mask. The urine samples were extracted with Solid Phase Extraction (SPE) cartridges and the compounds were separated and detected using Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS) due to their high sensitivity and selectivity in preventing most matrix interferences. The alpha- and beta-isomers of endosulfan were detected (total concentrations ranged between 5368 and 2239 pg/ml) but endosulfan-ether, -lactone or -sulphate were not found above the detection limits. The results obtained were compared with the concentrations found for a non-occupational exposed man. Statistical interpretation of the excretion-rate of the insecticide was performed by assuming that it can be described as a first-order kinetic. The constant rate and the half-lives were determined.

[Efficacy of the APimicos-B to control and prevent chalkbrood disease in honey bees]. / Estudio de la eficacia del APimicos-B en el control y la prevención de la ascosferiosis en la abeja de la miel.

Chalkbrood disease in Apis mellifera is a fungal disease affecting developing brood, infested larvae become mummified. As it is a factorial disease, studies on this pathology are obstructed by the need of some predisposing conditions which must occur for such disease to develop. Thus, many questions are yet to be answered about which treatments to apply. The aim of this work is to evaluate the efficacy of the Apimicos-B, a treatment against chalk brood. To induce the disease, some pieces of combs containing susceptible worker brood both from infected and treated colonies and from infected and untreated colonies were cooled. No significant differences were registered (53.12% and 59.58% of mummification respectively).

Analysis of acetamiprid in vegetables using gas chromatography-tandem mass spectrometry.

A new analytical method has been validated for determining the insecticide acetamiprid in vegetables using gas chromatography (OC) and different mass spectrometric detection techniques, such as full-scan mass spectrometry (MS), and tandem mass spectrometry (MS/MS). For this purpose, a previous extraction of the vegetable sample was carried out with ethyl acetate. In GC-MS/MS, the lowest detectable concentration was 0.001 mg kg(-1), the average recovery rates at various fortification levels (0.015 and 0.030 mg kg(-1)) ranged between 82.4 and 85.7% and the relative standard deviations were lower than 12.2% in all cases.

Evaluation of endosulfan residues in vegetables grown in greenhouses.

The reduction in residue levels of endosulfan with time after treatment of tomatoes, green beans, peppers and cucumbers grown in different types of commercial greenhouses (flat- and asymmetric-roof greenhouses) in Almería (Spain) was investigated. A study of the major and minor degradation products of endosulfan in peppers and cucumbers (endosulfan-sulfate, -ether and -lactone) was carried out using gas chromatography-tandem mass spectrometry (GC-MS/MS). To establish the influence of environmental conditions on the degradation of endosulfan, several field trials have been carried out in which crops were sprayed at different rates (full, half- and quarter- of those rates recommended) during two seasons (spring and winter). For statistical purposes, the disappearance of endosulfan with time was considered to follow a pseudo-first-order reaction. Analysis of variance (ANOVA) has been applied to the results obtained. Half-lives of residue disappearance were 4.03-4.68 days in green beans, 4.03-4.20 days in tomatoes, 8.22 days in peppers and 7.97 days in cucumbers. Half-lives in spring were shorter than in the winter season. The application rate and the type of greenhouse did not influence the half-lives.

Analysis of endosulfan and its metabolites in human serum using gas chromatography-tandem mass spectrometry.

A new analytical method combining solid-phase extraction and gas chromatography-tandem mass spectrometry (GC-MS-MS) is proposed to determine the insecticide endosulfan as well as its metabolites endosulfan-ether, -lactone, and -sulfate in human serum. Most matrix interferences are avoided using a cleanup step included in the sample treatment and an instrumental technique such as GC-MS-MS, which presents a high sensitivity and selectivity. Recoveries of spiked compounds range between 94.8 and 100.4% and 93.4 and 99.7% at fortification levels of 10 and 30 ng/mL, respectively. The relative standard deviation is lower than 17.6% in all cases, and the limits of detection calculated range from 6 to 19 pg/mL. Serum samples of nine agricultural workers that spray endosulfan into greenhouses in Almería (Spain) and two nonoccupationally exposed people are analyzed, and endosulfan is found in all the samples studied.

A comparative study of the sperm nuclear morphometry in cattle, goat, sheep, and pigs using a new computer-assisted method (CASMA-F).

This study was designed to compare the sperm nuclear morphometry of four species of domestic artiodactyls (cattle, sheep, goats, and pigs), using the newly developed automatic computer-assisted sperm morphometry analysis-F. The study was divided into two experiments. In the first experiment, samples from 20 males from each species were collected, diluted, and divided into four sample aliquots. The first was labeled directly with Hoechst 33342, and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying, and the other was fixed either with glutaraldehyde (GLUT), or with methanol, and afterward labeled with Hoechst. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and at least 200 sperm cells per sample were processed using the Image J analysis open software. Air-drying significantly reduced nuclear sperm dimensions in ruminant species, whereas no effect was observed in pigs. For most of the primary morphometric parameters, the relationship between the four species for the sperm nuclear dimensions can be described as follows: bull > ram ≥ boar > goat. However, ram sperm nuclei had greater width than those of the other species studied. For the secondary morphometric parameters, ram sperm nuclei were clearly less elliptical and elongated and showed greater regularity than in the other studied species. In the second experiment, ejaculates from 10 males per species were used to compare the sperm head morphometric results obtained with the computer-assisted sperm morphometry analysis-F system (using the GLUT treatment as reference) to a more conventional CASMA method (semen smears stained with Harris's hematoxylin and processed with the Integrated Sperm Analysis System [ISAS] commercial software [Proiser R&D SL, Buñol, Spain]). Spermatozoa displayed a bigger size when processed with Harris's hematoxylin than with the GLUT method in all primary sperm head morphometric parameters for the four species studied. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters in the four species studied. It was concluded that drying and fixation has little effect on sperm nuclear morphometry, with differences between species, and that there are significant variations in size of the sperm nucleus and in the hydrodynamic properties between the four species studied.