Metabolic Analysis of Schizochytrium Mutants With High DHA Content Achieved With ARTP Mutagenesis Combined With Iodoacetic Acid and Dehydroepiandrosterone Screening

High DHA production cost caused by low DHA titer and productivity of the current Schizochytrium strains is a bottleneck for its application in competition with traditional fish-oil based approach. In this study, atmospheric and room-temperature plasma with iodoacetic acid and dehydroepiandrosterone screening led to three mutants, 6–8, 6–16 and 6–23 all with increased growth and DHA accumulations. A LC/MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle might be relevant to the increased growth and DHA biosynthesis in the mutants. Finally, the mutant 6–23, which achieved the highest growth and DHA accumulation among all mutants, was evaluated in a 5 L fermentor. The results showed that the DHA concentration and productivity in mutant 6–23 were 41.4 g/L and 430.7 mg/L/h in fermentation for 96 h, respectively, which is the highest reported so far in literature. The study provides a novel strain improvement strategy for DHA-producing Schizochytrium.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Purification and characterization of thermo-alkaline stable lipase from Bacillus coagulans and its compatibility with commercially available detergents

Thermo-alkaline stable lipase producing B. coagulans was isolated and identified by 16S rRNA sequencing. The lipase production was optimized using different growth parameters. The enzyme was purified and characterized in terms of pH, temperature, solvents, heavy metal ions and inhibitors. Compatibility with commercially available detergents was also studied. The isolate showed maximum lipase production at 37 ⁰C; pH of 9 within 48 h. Addition of magnesium chloride increased lipase production. Sephadex G-100 chromatography was used to purify lipase. The enzyme showed maximum activity at pH 8 of 30 ⁰C. Lipase form B. coagulans was active at a wide range of temperature between 30-70 ⁰C. It was stable in most of the solvents at a concentration 5 and 10%, except dimethyl sulfoxide (DMSO) and methanol. Iodoacetic acid (IAA) and p-chloromercuribenzoic acid (pCMB) had an inhibitory effect on lipase. The lipase was compatible with commercially available detergents that increased the brightness and whiteness of the tested cotton fabrics. Lipase from B. coagulans with alkaline stability at a wide range of temperature has potential application in the detergent industry.

Iodoacetic acid, a water disinfection byproduct, disrupts hypothalamic and pituitary reproductive regulatory factors and induces toxicity in the female pituitary

Iodoacetic acid (IAA) is a water disinfection byproduct (DBP) formed by reactions between oxidizing disinfectants and iodide. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. In humans, DBPs have been epidemiologically associated with reproductive dysfunction. In mouse ovarian culture, IAA exposure significantly inhibits antral follicle growth and reduces estradiol production. Despite this evidence, little is known about the effects of IAA on the other components of the reproductive axis: the hypothalamus and pituitary. We tested the hypothesis that IAA disrupts expression of key neuroendocrine factors and directly induces cell damage in the mouse pituitary. We exposed adult female mice to IAA in drinking water in vivo and found 0.5 and 10 mg/L IAA concentrations lead to significantly increased mRNA levels of kisspeptin (Kiss1) in the arcuate nucleus, while not affecting Kiss1 in the anteroventral periventricular nucleus. Both 10 mg/L IAA exposure in vivo and 20 μM IAA in vitro reduced follicle stimulating hormone (FSHβ)-positive cell number and Fshb mRNA expression. IAA did not alter luteinizing hormone (LHβ) expression in vivo, though exposure to 20 μM IAA decreased expression of Lhb and glycoprotein hormones, alpha subunit (Cga) mRNA in vitro. IAA also had toxic effects in the pituitary, inducing DNA damage and P21/Cdkn1a expression in vitro (20 μM IAA) and DNA damage and Cdkn1a expression in vivo (500 mg/L). These data, implicate IAA as a hypothalamic-pituitary-gonadal axis toxicant and suggest the pituitary is directly affected by IAA exposure.

Effect of Tobacco on Knee Joint Recovery of College Students After Sports

Objectives: By analyzing the protective effect and mechanism of tobacco on knee joint cartilage in rats, this paper studies the effect of tobacco on knee joint recovery of college students after sports. Methods: Firstly, the main subunits of nAChRs were systematically studied by using the rat knee arthritis model α 7 and α 4 and β To clarify the correlation between nAChRs and the occurrence and development of OA. Then, the OA rat model prepared by iodoacetic acid was used as the experimental object to observe the protective effect of nicotine on knee osteoarthritis cartilage in rats. Results: The histological changes of rats in MIA group were obvious after operation. The results of light microscope score and Mankin's score at 15 and 30 days were significantly higher than those in con group. Of right knee cartilage in rats in MIA group α 7, α 4 and β The expression of 2 did not change significantly on the 15th day, but increased significantly on the 30th day compared with the blank control group. Conclusion: Nicotine has a protective effect on knee bone and joint cartilage and promotes the accelerated recovery of knee bone and joint after exercise.. Key words: nicotine, knee joint, cartilage, recovery after exercise.

Iodoacetic acid affects Estrous Cyclicity, ovarian gene expression, and hormone levels in mice

Iodoacetic acid (IAA) is a water disinfection byproduct that is an ovarian toxicant in vitro. However, information on the effects of IAA on ovarian function in vivo was limited. Thus, we determined whether IAA exposure affects estrous cyclicity, steroidogenesis, and ovarian gene expression in mice. Adult CD-1 mice were dosed with water or IAA (0.5–500 mg/L) in the drinking water for 35–40 days during which estrous cyclicity was monitored for 14 days. Ovaries were analyzed for expression of apoptotic factors, cell cycle regulators, steroidogenic factors, estrogen receptors, oxidative stress markers, and a proliferation marker. Sera were collected to measure pregnenolone, androstenedione, testosterone, estradiol, inhibin B, and follicle-stimulating hormone (FSH) levels. IAA exposure decreased the time that the mice spent in proestrus compared to control. IAA exposure decreased expression of the pro-apoptotic factor Bok, the cell cycle regulator Ccnd2, and borderline decreased expression of the anti-apoptotic factor Bcl2l10, the pro-apoptotic factor Aimf1, and the steroidogenic factor Cyp19a1 compared to control. IAA exposure increased expression of the pro-apoptotic factors Bax and Aimf1, the anti-apoptotic factor Bcl2l10, the cell cycle regulators Ccna2, Ccnb1, Ccne1, and Cdk4, and estrogen receptor Esr1 compared to control. IAA exposure decreased expression of Cat and Sod1, and increased expression of Cat, Gpx, and Nrf2. IAA exposure did not affect expression of Star, Cyp11a1, Cyp17a1, Hsd17b1, Hsd3b1, Esr2 or Ki67 compared to control. IAA exposure decreased estradiol levels, but did not alter other hormone levels compared to control. In conclusion, IAA exposure alters estrous cyclicity, ovarian gene expression, and estradiol levels in mice.

Exposure to Iodoacetic Acid, a Water Disinfection Byproduct, Leads to Abnormal Expression of Key Reproductive Axis Genes in the Hypothalamus and Pituitary

Iodoacetic acid (IAA) – a water disinfection byproduct (DBP) formed from the reaction between an oxidizing disinfectant, i.e. chlorine, and iodide – is an understudied, yet potentially dangerous environmental toxicant. DBPs have been epidemiologically associated with reproductive dysfunction. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. Further, murine ovarian research has shown that IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels. Despite this evidence, little is known about the other components of the reproductive axis: the hypothalamus and pituitary. To address this, we tested the hypothesis that IAA exposure would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We exposed adult female CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water from postnatal day 40 (P40) to their first day in diestrus after P75. From this experiment, we collected whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA), and processed them for mRNA analysis. We also exposed pituitary explant cultures to IAA to observe direct effects on gene expression. In vivo, we found that mRNA levels of kisspeptin (Kiss1) are significantly increased in the ARC, the region that controls pulsatile GnRH release, at 0.5 and 10 mg/L IAA concentrations. Kiss1 is unchanged in the AVPV, the neuron population responsible for generating the LH surge. We also measured ARC expression of neurokinin B (Tac2) and dynorphin (Pdyn), neuropeptides secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We saw no difference in either. GnRH (Gnrh1) expression was also unchanged. Both in vivo at 10 mg/L IAA and in culture, we found IAA exposure significantly reduced Fshb mRNA. Preliminary immunohistochemistry (IHC) data suggests it also leads to an apparent reduction in FSH-positive cells in vitro (N=2). Lhb and the α-subunit (Cga) were unaltered in vivo, though were significantly reduced with in vitro exposure. In neither context was mRNA expression of the GnRH receptor (Gnrhr) changed. Noting apparent direct effects of IAA on the pituitary, we assessed expression of the cell-cycle inhibitor p21 (Cdkn1a), which has been shown to increase with toxicant exposure. We found Cdkn1a increased in vivo at 500 mg/L IAA, trending at 100 mg/L (p=.070), and in vitro. IHC data in vitro suggests a marked increase in P21-positivity following IAA exposure. These data, together with prior ovarian findings, implicate IAA as a potential reproductive axis disruptor at each major level – through ARC Kiss1 expression, Fshb expression in vivo and in vitro, FSH expression in vitro, and Lhb and Cga in vitro. Further, Cdkn1a/P21 induction indicates IAA toxicity at the level of the pituitary.

Antibacterial Mechanisms of Orostachys Cartilaginous Cell Cultures: Effect on Cell Permeability and Respiratory Metabolism of Bacillis Subtilis

Orostachys cartilaginous of Crassulaceae family is a plant native to the Changbai Mountain area, China. Although O. cartilaginous has various medicinal values, its product development and production are restricted by the insufficient resource available. O. cartilaginous cell cultures possess an efficient antibacterial effect against Bacillis subtilis, but the underlying mechanism is not clear yet. Therefore, this study investigated the effects of extract from bioreactor cultured O. cartilaginous cells (OE) on B. subtilis cell permeability and respiratory metabolism to provide a reference for the further utilization of O. cartilaginous cell cultures. Results showed alkaline phosphatase activity, electrical conductivity, nucleic acid and protein contents in B. subtilis suspensions were significantly increased (p < 0.01) by OE treatment, indicating the occurrence of cell damage or increase in cell permeability. OE inhibited B. subtilis respiration, and the combination groups of OE+iodoacetic acid (IA) and OE+sodium phosphate (SP) showed low superposition rates (approximately 35%), revealing that OE likely affected IA- and SP-represented metabolic pathways. The activities of B. subtilis enzymes, specifically, hexokinase and pyruvate kinase in the Embden-Meyerhof (EMP) pathway and glucose-6-phosphate dehydrogenase in the pentose phosphate (HMP) pathway, decreased after OE treatment. This result proved that OE inhibited B. subtilis respiration by regulating the EMP and HMP pathways.