Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150–200 nm) by Nanosight^® and electron microscopy. Zygotes were cultured in the presence of 3x10⁵EVs/mL, 1.5x10⁵EVs/mL or 7.5x10⁴EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7–9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.     

Aquaporin-1 expression in proximal tubule epithelial cells of human kidney is regulated by hyperosmolarity and contrast agents

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents.     

Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37

The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performances of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model as opposed to kinetic models. Using the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a "macromodeling" technique to rapidly develop dynamic models of a process.     

Bioconversion of 2α-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.     

Effects of ozone on photosynthetic CO2 exchange, chlorophyll a fluorescence and antioxidant systems in lettuce leaves

The effects of ozone exposure on lettuce leaves were investigated by means of gas exchange, modulated chlorophyll a fluorescence and antioxidants systems. High ozone concentration decreased the rate of photosynthesis at light saturation level and changes in stomatal conductance and transpiration rate. However, an increase in intercellular CO₂ concentration indicated a decrease in the carboxylation efficiency. These data agreed with a reduction of non-cyclic electron flow and lower capacity to reduce the quinone pool. Ozone affected the ascorbate pool and decreased ascorbate peroxidase activity, increased lipid peroxidation, altered membrane properties and reduced the development of non-photochemical quenching.