Improving Hypoxia Adaption Causes Distinct Effects on Growth and Bioactive Compounds Synthesis in an Entomopathogenic Fungus Cordyceps militaris.

Cordyceps militaris is an entomopathogenic fungus producing a variety of bioactive compounds. To meet the huge demand for medicinal and edible products, industrialized fermentation of mycelia and cultivation of stromata have been widely developed in China. The content of bioactive metabolites of C. militaris, such as cordycepin, is higher when cultivated on silkworm pupae than on rice or in broth. However, compared with other cultivation methods, C. militaris grows more slowly and accumulates less biomass. The hypoxic environment in pupa hemocoel is one of environmental factor which is not existed in other cultivation methods. It is suggested that hypoxia plays an important role on the growth and the synthesis of bioactive compounds in C. militaris. Here, we demonstrated that the distinct effects on the growth and synthesis of bioactive compounds employing different strategies of improving hypoxia adaption. The introduction of Vitreoscilla hemoglobin enhanced growth, biomass accumulation, and crude polysaccharides content of C. militaris. However, cordycepin production was decreased to 9-15% of the control group. Meanwhile, the yield of adenosine was increased significantly. Nonetheless, when the predicted bHLH transcription factor of sterol regulatory element binding proteins (SREBPs) was overexpressed in C. militaris to improve the hypoxia adaption of fungal cells, cordycepin content was significantly increased more than two-fold. These findings reveal the role of SREBPs on growth and bioactive compounds synthesis. And it also provides a scientific basis for rationally engineering strains and optimization strategies of air supply in cultivation and fermentation.

Alleviating product inhibition of Trichoderma reesei cellulase complex with a product-activated mushroom endoglucanase.

Product inhibition of cellulase is a challenging issue in industrial processes. Here, we introduced a product-activated mushroom cellulase, PaCel3A from Polyporus arcularius, into Trichoderma reesei. The filter paper activity, carboxymethyl cellulase activity, and saccharification efficiency (substrate: pretreated rice straw, PRS) of transformants increased significantly with this enzyme (by 18.4-26.8%, 13.8-22.8%, and 17.0%, respectively). A mutant of PaCel3A, PaCel3AM, obtained based on B-factor analysis, saturated mutagenesis, and residual activity assay, showed improved thermostability. The PRS saccharification efficiency using the cellulase complex from T. reesei transformants overexpressing pacel3am increased by 56.4%-63.0%. In addition, the T. reesei cellulase complex obtained by adding the purified recombinant PaCel3AM from T. reesei (rCel3aM-tr) to hydrolyze PRS resulted in increased reducing sugar yields at all sampling points, outperforming the cellulase complexes without rCel3aM-tr. These results suggest that introducing product-activated cellulase genes is a simple and feasible method to alleviate the product inhibition of cellulase.

The effects of pprI gene of Deinococcus radiodurans R1 on acute radiation injury of mice exposed to 60Co γ-ray radiation.

The role of the pprI gene from Deinococcus radiodurans R1 in therapy of acute radiation injury of a mammalian host was investigated. We injected a plasmid containing the pprI gene into the muscle of mice exposed to total 6Gy of 60Co γ-ray radiation. After injection, we used in vivo gene electroporation technology to transfer the pprI gene into the cell. We found the PprI protein was expressed significantly at 1 d after irradiation, but there was no expression of pprI gene 7 d post-irradiation. The expression of pprI gene evidently decreased the death rate of mice exposed to lethal dose radiation, significantly relieved effects on blood cells in the acute stage, shortened the persistence time of the decrease of lymphocytes, and decreased the apoptotic rates of spleen cells, thymocytes and bone marrow cells. The expression of Rad51 protein in the lungs, livers, and kidneys was significantly higher in the mice treated with the pprI plasmid after irradiation. However, there were no obvious differences for Rad52 protein expression. We conclude that the prokaryotic pprI gene of D. radiodurans R1 first was expressed in mammalian cells. The expressed prokaryotic PprI protein has distinct effects of the prevention and treatment on acute radiation injury of mammal. The effects of radio-resistance may relate to expression of Rad51 protein which is homologous with RecA from D. radiodurans.

MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/ß-catenin pathway.

A stem-like subpopulation existed in GBM cells, called glioma stem cells (GSCs), might contribute to cancer invasion, angiogenesis, immune evasion, and therapeutic resistance, providing a rationale to eliminate GSCs population and their supporting niche for successful GBM treatment. LincRNA-p21, a novel regulator of cell proliferation, apoptosis and DNA damage response, is found to be downregulated in several types of tumor. However, little is known about the role of lincRNA-p21 in stemness and radioresistance of GSCs and its regulating mechanisms. In this study, we found that lincRNA-p21 negatively regulated the expression and activity of ß-catenin in GSCs. Downregulation of lincRNA-p21 in GSCs was resulted from upregulation of Hu antigen R (HuR) expression caused by miR-146b-5p downregulation. MiR-146b-5p overexpression increased apoptosis and radiosensitivity, decreased cell viability, neurosphere formation capacity and stem cell marker expression, and induced differentiation in GSCs. Moreover, knock-down lincRNA-p21 or HuR and ß-catenin overexpression could rescue the phenotypic changes resulted from miR-146b-5p overexpression in GSCs. These findings suggest that targeting the miR-146b-5p/HuR/lincRNA-p21/ß-catenin signaling pathway may be valuable therapeutic strategies against glioma.

The protein PprI provides protection against radiation injury in human and mouse cells.

Severe acute radiation injuries are both very lethal and exceptionally difficult to treat. Though the radioresistant bacterium D. radiodurans was first characterized in 1956, genes and proteins key to its radioprotection have not yet to be applied in radiation injury therapy for humans. In this work, we express the D. radiodurans protein PprI in Pichia pastoris yeast cells transfected with the designed vector plasmid pHBM905A-pprI. We then treat human umbilical endothelial vein cells and BALB/c mouse cells with the yeast-derived PprI and elucidate the radioprotective effects the protein provides upon gamma irradiation. We see that PprI significantly increases the survival rate, antioxidant viability, and DNA-repair capacity in irradiated cells and decreases concomitant apoptosis rates and counts of damage-indicative γH2AX foci. Furthermore, we find that PprI reduces mortality and enhances bone marrow cell clone formation and white blood cell and platelet counts in irradiated mice. PprI also seems to alleviate pathological injuries to multiple organs and improve antioxidant viability in some tissues. Our results thus suggest that PprI has crucial radioprotective effects on irradiated human and mouse cells.

Deinococcus radiodurans pprI expression enhances the radioresistance of eukaryotes.

PprI accelerates radiation-induced DNA damage repair via regulating the expression of DNA repair genes and enhances antioxidative enzyme activity in Deinococcus radiodurans after radiation. The main aim of our study was to determine whether the expression of pprI gene could fulfil its DNA repair function in eukaryotes and enhance the radioresistance of eukaryotic organism or not. In this study, we constructed pEGFP-c1-pprI eukaryotic expression vector and established a human lung epithelial cell line BEAS-2B with stable integration of pprI gene. We found that pprIexpression enhanced radioresistance of BEAS-2B cells, decreased γ-H2AX foci formation and apoptosis in irradiated BEAS-2B cells and alleviated radiation induced G2/M arrest of BEAS-2B cells. Moreover, we transferred pEGFP-c1-pprI vector into muscle of BALB/c mice by in vivo electroporation and studied the protective effect of prokaryotic pprI gene in irradiated mice. We found that pprI expression alleviated acute radiation induced hematopoietic system, lung, small intestine and testis damage and increased survival rate of irradiated mice via regulating Rad51 expression in different organs. These findings suggest that prokaryotic pprI gene expression in mammalian cells could enhance radioresistance in vitro and in vivo.

Responses to Starch Infusion on Milk Synthesis in Low Yield Lactating Dairy Cows.

The effect of starch infusion on production, metabolic parameters and relative mRNA abundance was investigated in low yield lactating cows from 86 days in milk. Six Holstein cows fitted with permanent ruminal cannulas were arranged into one of two complete 3×3 Latin squares and infused with a starch solution containing 800 grams starch for 16 days. The three treatments were: i) ruminal and abomasal infusion with water (Control); ii) ruminal infusion with cornstarch solution and abomasal infusion with water (Rumen); iii) ruminal infusion with water and abomasal infusion with cornstarch solution (Abomasum). There were no significant differences (p>0.05) among the three treatments with low yield lactating cows in feed and energy intake, milk yield and composition, plasma metabolism, or even on gene expression. However, cows receiving starch through rumen performed better than directly through the abomasum during the glucose tolerance test procedure with a higher area under the curve (AUC; p = 0.08) and shorter half-time (t(1/2); p = 0.11) of plasma insulin, therefore, it increased glucose disposal, which stated a lipid anabolism other than mobilization after energy supplementation. In conclusion, extra starch infusion at concentration of 800 g/d did not enhance energy supplies to the mammary gland and improve the lactating performance in low yield lactating cows.

Changes in feed intake, nutrient digestion, plasma metabolites, and oxidative stress parameters in dairy cows with subacute ruminal acidosis and its regulation with pelleted beet pulp.

The objectives of this study were to 1) determine the variation of nutrient digestion, plasma metabolites and oxidative stress parameters triggered by induced subacute ruminal acidosis (SARA); and 2) evaluate the ability of pelleted beet pulp (BP) as a replacement for ground corn to alleviate SARA. Eight Holstein-Friesian cows were fed four diets during four successive17-day periods: 1) total mixed ration (TMR) containing 0% finely ground wheat (FGW) (W0); 2) TMR containing 10% FGW (W10); 3) TMR containing 20% FGW (W20); and 4) TMR containing 10% BP as a replacement for 10% ground corn (BP10). The SARA induction protocol reduced the mean ruminal pH from 6.37 to 5.94, and the minimum ruminal pH decreased from 5.99 to 5.41 from baseline to challenge period. Mean ruminal pH increased from 5.94 to 6.05, and minimum daily ruminal pH increased from 5.41 to 5.63, when BP was substituted for corn. The apparent digestibility of nutrients was not affected by the dietary treatments, except that the digestibility of neutral detergent fibre (NDF) and acid detergent fibre (ADF) was reduced in cows fed the W20 diet compared with cows fed the W0 and W10 diets, and cows fed the BP10 diet had higher NDF and ADF digestibility than the cows fed the W20 diet. Cows fed the W20 diet had a lower plasma concentration of ß-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA), cholesterol, triglyceride, and total antioxidative capacity (TAC), and a higher plasma concentration of glucose, insulin, malonaldehyde (MDA), super oxygen dehydrogenises (SOD), and glutathione peroxidase (GSH-Px) than cows fed the W0 diet. Substitution of BP for corn increased concentrations of plasma BHBA and TAC, but decreased concentrations of plasma MDA. Our results indicate that reduction of fibre digestion; the concomitant increase of plasma glucose and insulin; the decrease of plasma BHBA, NEFA, cholesterol, and triglyceride; and changes of plasma oxidative stress parameters are highly related to SARA induced by W20 diets. These variables may be alternative candidates for SARA diagnosis. We also suggest that the substitution of BP for corn could reduce the risk of SARA, increase fibre digestion, and improve the antioxidant status in dairy cows.

Long non-coding RNA GAS5 functions as a tumor suppressor in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is a malignancy with a poor prognosis. We aimed to explore whether the expression of Long Non-Coding RNA (LncRNA) growth arrest-specific transcript 5 (GAS5) is associated with RCC genesis. METHODS: We selected twelve clinical samples diagnosed for renal clear cell carcinoma and found that the LncRNA GAS5 transcript levels were significantly reduced relative to those in adjacent unaffected normal renal tissues. RESULTS: In addition, expression of GAS5 was lower in the RCC cell line A498 than that in normal renal cell line HK-2. Furthermore, using functional expression cloning, we found that overexpression of GAS5 in A498 cells inhibited cell proliferation, induced cell apoptosis and arrested cell cycling. At the same time, the migration and invasion potential of A498 cells were inhibited compared to control groups. CONCLUSION: Our study provided the first evidence that a decrease in GAS5 expression is associated with RCC genesis and progression and overexpression of GAS5 can act as a tumor suppressor for RCC, providing a potential attractive therapeutic approach for this malignancy.