Optimizing Decellularization of Bovine Ovarian Tissue: Toward a Transplantable Artificial Ovary Scaffold with Minimized Residual Toxicity and Preserved Extracellular Matrix Morphology.

INTRODUCTION: The decellularized extracellular matrix (dECM) from ovarian tissue could be the best scaffold for the development of a transplantable artificial ovary. Typically, dECM from ovarian tissue has been obtained using sodium dodecyl sulfate (SDS), at a concentration of 1% for 24 h. However, SDS can leave residues in the tissue, which may be toxic to the seeded cells. This study aimed to obtain dECM from bovine ovarian tissue using SDS and NaOH at a minimum concentration in the shortest incubation time. METHODS: The respective SDS and NaOH concentrations investigated were 1% and 0.2M; 0.5% and 0.1M; 0.1% and 0.02M, and 0.05% and 0.01M, with 24, 12, and 6 h incubation periods. After the incubation time the tissue was washed in 50 mL of distilled water for 6 h. RESULTS: Histological analysis confirmed decellularization and showed the conservation of collagen fibers in all samples following treatment. Furthermore, the lowest SDS and NaOH concentrations that showed no DNA remaining during electrophoresis analysis were 0.1% and 0.02M when incubated for 24 and 12 h. DNA quantification resulted in ˂ 0.2 ng DNA/mg ovarian tissue using these protocols. Additionally, the coculture of dECM (obtained by 0.1% SDS and 0.02M NaOH for 12 h) with ovarian cells showed that there was no toxic effect for the cells for up to 72 h. CONCLUSION: The protocol involving 0.1% SDS and 0.02M NaOH for 12 h incubation decellularizes bovine ovarian tissue, generating a dECM that preserves the native ECM morphology and is non-toxic to ovarian cells.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

Differential metabolism of Mycoplasma species as revealed by their genomes

The annotation and comparative analyses of the genomes of Mycoplasma synoviae and Mycoplasma hyopneumonie, as well as of other Mollicutes (a group of bacteria devoid of a rigid cell wall), has set the grounds for a global understanding of their metabolism and infection mechanisms. According to the annotation data, M. synoviae and M. hyopneumoniae are able to perform glycolytic metabolism, but do not possess the enzymatic machinery for citrate and glyoxylate cycles, gluconeogenesis and the pentose phosphate pathway. Both can synthesize ATP by lactic fermentation, but only M. synoviae can convert acetaldehyde to acetate. Also, our genome analysis revealed that M. synoviae and M. hyopneumoniae are not expected to synthesize polysaccharides, but they can take up a variety of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). Our data showed that these two organisms are unable to synthesize purine and pyrimidine de novo, since they only possess the sequences which encode salvage pathway enzymes. Comparative analyses of M. synoviae and M. hyopneumoniae with other Mollicutes have revealed differential genes in the former two genomes coding for enzymes that participate in carbohydrate, amino acid and nucleotide metabolism and host-pathogen interaction. The identification of these metabolic pathways will provide a better understanding of the biology and pathogenicity of these organisms.