Effects of HMGA2 on the epithelial-mesenchymal transition-related genes in ACHN renal cell carcinoma cells-derived xenografts in nude mice.

BACKGROUND: The architectural transcriptional regulator high-mobility group AT-hook 2 (HMGA2) is an oncofetal protein which has been reported to be ectopically expressed in a variety of cancers. A high expression of HMGA2 in human renal cell carcinoma (RCC) is related with tumor invasiveness and poor prognosis. Recent in vitro studies have shown that HMGA2 knockdown was able to decrease cell proliferation and migration, and regulate the gene expression related to epithelial-mesenchymal transition (EMT). METHODS: To understand the HMGA2's effect in vivo, HMGA2 expression was knocked down in ACHN cells using small hairpin RNA (shRNA), then the HMGA2-deficient ACHN cells were xenografted into the BALB/c nude mice. Tumor growth was monitored and the expression of EMT-related genes was analyzed. RESULTS: HMGA2 expression was confirmed to be knocked down in the cultured and xenografted ACHN cells. The xenograft tumor of HMGA2-deficient cells demonstrated a retarded growth pattern compared with the control. The expression of E-cadherin was increased, whereas N-cadherin and Snail were decreased in the HMGA2-deficient xenograft tumors. CONCLUSIONS: In conclusion, to the best of our knowledge, for the first time, we have successfully developed an in vivo experiment using HMGA2-silencing ACHN cells to be grown as xenografts in nude mice. Our findings show that HMGA2 deficiency was sufficient to suppress the xenograft tumor growth in vivo, which support our hypothesis that HMGA2-induced renal carcinogenesis occurs at least in part through the regulation of tumor associated EMT genes.

A PDMS-based microneedle array electrode for long-term ECG recording.

To acquire high-quality electrocardiogram (ECG) signals, traditional Ag/AgCl wet electrodes used together with conductive gel can effectively reduce electrode-skin interface impedance (EII) in a short term. However, their weaknesses of poor flexibility and instability can no longer meet the long-term monitoring requirements of intelligent wearable devices. Owing to the flexible dry electrode without conductive gel, it is a good choice to solve the critical problem on drying-out of conductive gel. Therefore, we develop a flexible microneedle array electrode (FMAE) based on polydimethylsiloxane (PDMS) substrate, which obtains reliable bioelectrical signals by way of penetrating into the stratum corneum (SC) of the skin. The fabrication process, including silicon mold, twice PDMS shape-transferring and encapsulation, has advantages of low cost, repeatable production and good biocompatibility. Afterwards, by comparing the performance with different electrodes, impedance test results indicate that the impedance of FMAE are smaller and more stable, and ECG tests in long term and at resting/jogging states also verify that FMAE can obtain durable, stable and reliable signals. In conclusion, FMAE is promising in long-term ECG monitoring.

Enhancement of adenosine production by Bacillus subtilis CGMCC 4484 through metabolic flux analysis and simplified feeding strategies.

The objective of this research was to understand how the initial glucose concentration influences adenosine (AR) production and metabolic flux shift on the cultivation of Bacillus subtilis CGMCC 4484. Experiments confirmed that initial glucose concentration affects cell growth, AR production and metabolites, significantly. The flux distribution at the key nodes of glucose-6-phosphate (G6P), pyruvate (PYR) and acetyl coenzyme-A (AcCoA) could be affected by changing the glucose concentration. Based on kinetic analysis of specific rates, the low-glucose concentration was better for both cell growth and AR production during the first 12 h. However, the high-glucose concentration was more favorable for AR formation after 18 h. Furthermore, different simplified feeding strategies were designed to achieve higher AR accumulation. The final AR concentration of 15.60 g L(-1) was achieved when an optimized constant-feeding strategy was used, which was 21.02 % higher than batch fermentation. This was the first time to investigate the regulation of the glucose metabolism of AR-producing B. subtilis.

Artemisitene Alters LPS-Induced Oxidative stress, inflammation and Ferroptosis in Liver Through Nrf2/HO-1 and NF-kB Pathway.

The liver plays a critical role in sepsis, which is a serious worldwide public health problem. A novel mechanism of controlled cell death called ferroptosis has recently been described. Disrupted redox equilibrium, excessive iron, and enhanced lipid peroxidation are key features of ferroptosis. It is unknown how ferroptosis affects liver damage caused by sepsis. In the present study, we aimed to elucidate the pathways and explore the impact of artemisitene (ATT) on ferroptosis in sepsis-induced liver injury. Our findings demonstrated that ATT significantly decreased liver damage and ferroptotic characteristics. Additionally, ATT significantly reduced the expression of the nuclear factor-κB (NF-κB) subunit to reduce LPS-induced hepatic oxidative stress and inflammation and upregulated the expression of nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream protein heme oxygenase 1 (HO-1). This may offer a new strategy for preventing LPS-induced hepatic injury.

Cloning, expression, and characterization of an adenylate cyclase from Arthrobacter sp. CGMCC 3584.

The cya gene encoding adenylate cyclase was cloned from Arthrobacter sp. CGMCC 3584 by thermal asymmetric interlaced PCR for the first time. It exhibited an open reading frame containing 1,125 bp and encoding 374 amino acids. Amino acid sequence analysis showed that this enzyme was a class III adenylate cyclase. Expression of the cya gene was carried out in Escherichia coli Rosetta, and purification was performed via Ni2+-NTA agarose gel column. SDS-PAGE indicated that the molecular mass of the recombinant adenylate cyclase was 45 kDa. The V max and K m were determined to be 5.06 µmol/min/mg and 7.56 mM, respectively. The optimum pH and temperature were 8.0 and 35 °C. Several divalent metal ions were found to activate the enzyme to different extents, and the maximal specific activity reached 3.04 µmol/min/mg when 50 mM Mg2+ was added. This was the first report of the cloning of an adenylate cyclase gene from Arthrobacter sp.

Bi-stage control of dissolved oxygen to enhance cyclic adenosine monophosphate production by Arthrobacter A302.

Experiments confirmed dissolved oxygen (DO) definitely affects cyclic adenosine monophosphate (cAMP) production by Arthrobacter A302. Production of cAMP by batch fermentation was investigated under various DO conditions. A two-stage DO control strategy was proposed to achieve optimal production of cAMP based on the kinetic analysis: the DO level was controlled at 40% during the first 18 h and then switched to 30%. Relatively high cAMP production (9.9 g L(-1)) was achieved by applying this strategy. The cAMP productivity (0.14 g L(-1) h(-1)) was also successfully improved by 85.1, 59.3, 15.1 and 28.0%, compared to cases in which DO was uncontrolled or DO levels were held at 20, 30 and 40%, respectively. This is the first report of the use of a two-stage DO control strategy in cAMP production, and it was verified to be an effective method for enhancing the cAMP yield via this strain.

Kinetic models of ribonucleic acid fermentation and continuous culture by Candida tropicalis no.121.

During ribonucleic acid fermentation, the fermentative processes were researched at pH controlled at 4.0 and under natural conditions. Unstructured models in a 50-L airlift fermentor were established for batch RNA production at pH 4.0 using the Verhulst equation for microbial growth, the Luedeking-Piret equation for product formation and a Luedeking-Piret-like equation for substrate uptake. Parameters of the kinetic models were determined using origin 7.5. Based on the models estimated above, another batch fermentation experiment was conducted in a 300-L airlift fermentor, which demonstrated that the models could be useful for RNA production on an industrial scale. Additionally, continuous fermentation based on kinetic models was proposed to make full use of substrates and reduce the cost of waste water treatment. As a result, although the DCW and RNA concentration were 11.5 and 1.68 g L(-1), which were lower than that of batch fermentation, the sugar utilization increased by 14.3%, while the waste water decreased by more than 90%.

Enhanced uridine 5'-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux.

A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

Production of cyclic adenosine monophosphate by Arthrobacter sp. A302 using fed-batch fermentation with pH-shift control.

The production of cyclic adenosine monophosphate (cAMP) by Arthrobacter sp. A302 was studied in a 5 L stirred tank fermentor under a range of pH values (6.5-8.0) and glucose feeding rates. In batch fermentation under a controlled pH, the optimum pH for cell growth was 7.5 with dry cell density (X) of 11.43 g L, and the optimum pH for cAMP accumulation was 7.0 with cAMP concentration of 7.41 g L. In order to achieve the high X and cAMP yield simultaneously, a pH-shift control strategy was proposed based on kinetic analysis of specific cell growth rate (µ) and specific cAMP formation rate (q ( s )). In this method, pH was controlled to 7.0 for the first 30 h of fermentation, and then subsequently shifted to 7.5 and maintained until the end of the process. Application of this approach significantly enhanced the cAMP concentration. Thereafter, cAMP production was further improved by combining the above-mentioned pH-control system and fed-batch process with glucose at a constant feeding rate of 1.0 g L(-1 )h(-1). Under optimum conditions, the final cAMP production was 10.87 g L, which is 110.0, 46.7, and 27.7% higher than that of the pH-uncontrolled, pH-controlled, and pH-shift controlled methods, respectively.

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5'-monophosphate by Saccharomyces cerevisiae.

A whole cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l(-1) sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.

Enhanced uridine diphosphate N-acetylglucosamine production using whole-cell catalysis.

Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5'-monophosphate (5'-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29 degrees C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5'-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%.

Optimization and validation of a GC-FID method for the determination of acetone-butanol-ethanol fermentation products.

An improved, simple gas chromatography-flame ionization detection (GC-FID) method was developed for measuring the products of acetone-butanol-ethanol (ABE) fermentation and the combined fermentation/separation processes. The analysis time per sample was reduced to less than 10 min compared to those of a conventional GC-FID (more than 20 min). The behavior of the compounds in temperature-programmed gas chromatographic runs was predicted using thermodynamic parameters derived from isothermal runs. The optimum temperature programming condition was achieved when the resolution for each peak met the analytical requirement and the analysis time was shortest. With the exception of acetic acid, the detection limits of the presented method for various products were below 10 mg/L. The repeatability and intermediate precision of the method were less than 10% (relative standard deviation). Validation and quantification results demonstrated that this method is a sensitive, reliable and fast alternative for conventional investigation of the adsorption-coupled ABE fermentation process.

Effect of ribose, xylose, aspartic acid, glutamine and nicotinic acid on ethyl (S)-4-chloro-3-hydroxybutanoate synthesis by recombinant Escherichia coli.

Most reductases which belong to the short chain dehydrogenase/reductase (SDR) superfamily require NAD (P) H for activity. Addition of this cofactor was still necessary for the production of ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli even when a cofactor regeneration system was constructed by co-expressing carbonyl reductase from Pichia stipitis (PsCRI) and glucose dehydrogenase from Bacillus megaterium (BmGDH). In an attempt to reduce dependence on the expensive cofactor, compounds directly or indirectly involved in NADP synthesis were added to the medium. Only glutamine and xylose enhanced the content of intracellular NADP (H) and the concentration of product. The concentration and yield of (S)-CHBE reached 730 mM and 48.7%, with 30 g/L of glutamine and 40 g/L of xylose, a 2.6-fold increase over the control without the addition of the two compounds.

Ion-exclusion chromatography determination of organic acid in uridine 5'-monophosphate fermentation broth.

Simultaneous determination of organic acids using ion-exclusion liquid chromatography and ultraviolet detection is described. The chromatographic conditions are optimized when an Aminex HPX-87H column (300 × 7.8 mm) is employed, with a solution of 3 mmol/L sulfuric acid as eluent, a flow rate of 0.4 mL/min and a column temperature of 60°C. Eight organic acids (including orotic acid, α-ketoglutaric acid, citric acid, pyruvic acid, malic acid, succinic acid, lactic acid and acetic acid) and one nucleotide are successfully quantified. The calibration curves for these analytes are linear, with correlation coefficients exceeding 0.999. The average recovery of organic acids is in the range of 97.6% ∼ 103.1%, and the relative standard deviation is in the range of 0.037% ∼ 0.38%. The method is subsequently applied to obtain organic acid profiles of uridine 5'-monophosphate culture broth fermented from orotic acid by Saccharomyces cerevisiae. These data demonstrate the quantitative accuracy for nucleotide fermentation mixtures, and suggest that the method may also be applicable to other biological samples.

Adaptation of glycolysis and growth to acetate in Sporolactobacillus sp. Y2-8.

Exogenous addition of a low concentration of acetate (2 g/L sodium acetate) effectively decreased acetic acid excretion and lowered the ATP content in Sporolactobacillus sp. Y2-8 without any growth defect although the acetate could not be utilized at an initial glucose concentration of 150 g/L. This induced an enhanced glycolytic flux with increased specific activities of hexokinase and phosphofructokinase, probably to compensate for the lowered efficiency of ATP production. However, with increasing concentrations (5 g/L sodium acetate), acetate was utilized first before being produced again, causing a growth lag at the transition. Glucose consumption was also reduced at high acetate concentrations, resulting in decreased D-lactic acid production. These results demonstrate that acetate plays a significant role in regulating glycolysis and growth of Sporolactobacillus.

Selective separation of biobutanol from acetone-butanol-ethanol fermentation broth by means of sorption methodology based on a novel macroporous resin.

The traditional distillation method for recovery of butanol from fermentation broth is an energy-intensive process. Separation of butanol based on adsorption methodology has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. However, the application of the commercial adsorbents in the integrated acetone-butanol-ethanol (ABE) fermentation process is restricted due to the low recovery (less than 85%) and the weak capability of enrichment in the eluent (3-4 times). In this study, we investigated the sorption properties of butanol onto three kinds of adsorbents with different polarities developed in our laboratory, that is, XD-41, H-511, and KA-I resin. The sorption behaviors of single component and ABE ternary mixtures presented in the fermentation broths on KA-I resin were investigated. KA-I resin had higher affinity for butanol than for acetone, ethanol, glucose, acetic acid, and butyric acid. Multicomponent ABE sorption on KA-I resin was modeled using a single site extended Langmuir isotherm model. In a desorption study, all the adsorbed components were desorbed in one bed volume of methanol, and the recovery of butanol from KA-I resin was 99.7%. The concentration of butanol in the eluent was increased by a factor of 6.13. In addition, KA-I resin was successfully regenerated by two bed volumes of water. Because of its quick sorption, high sorption capacity, low cost, and ease of desorption and regeneration, KA-I resin exhibits good potential for compatibility with future ABE fermentation coupled with in situ recovery product removal techniques.

Production of cytidine 5'-diphosphorylcholine with high utilization of ATP by whole cells of Saccharomyces cerevisiae.

Cytidine 5'-diphosphorylcholine (CDP-choline) was produced using a high efficiency ATP regeneration system and the Kennedy pathway in whole cells of Saccharomyces cerevisiae As 2.398. Out of eight variables, KH(2)PO(4), glycerol and (NH(4))(2)SO(4) were considered to be the most significant factors by response surface methodology including a Plackett-Burman design, path of steepest accent and central composite design. The optimum levels of the three variables were 20.13g/L KH(2)PO(4), 12.35g/L glycerol and 0.49g/L (NH(4))(2)SO(4), respectively. Energy utilization efficiency increased from 10.59% to 16.72% and choline chloride conversion yields increased from 12.35% to 42.78%. A high efficiency ATP regeneration system improves CDP-choline production.

Enhanced cyclic adenosine monophosphate production by Arthrobacter A302 through rational redistribution of metabolic flux.

Cyclic adenosine monophosphate (cAMP) was synthesized through the purine salvage synthesis pathway by Arthrobacter A302. Results showed that hypoxanthine was the best of the precursors, and the cAMP concentration reached 4.06 g/L. For inhibition of the glycolytic pathway, sodium fluoride was found the optimal effector, which was further studied on cAMP production. With the addition of 0.4 g/L of sodium fluoride, the maximal cAMP concentration reached 11.04 g/L, and the concentrations of lactic acid, alpha-ketoglutarate and citric acid were decreased by 77%, 86% and 76%, respectively. Meanwhile, the specific activities of glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase were decreased by 66%, 61%, and 46%, respectively. By contrast the activity of 6-phosphoglucose dehydrogenase was increased by 100%, which demonstrated the redistribution of metabolic flux. This is the first study to reveal the regulatory mechanisms of different effectors on cAMP production among the EMP pathway, HMP pathway and TCA cycle.

Effect of NH4+ and glycerol on cytidine 5'-diphosphocholine synthesis in Saccharomyces cerevisiae.

Both stimulation of ammonium ion on the glycolytic flux and regulation by glycerol of enzymes in Kennedy pathway for cytidine diphosphate choline production in S. cerevisiae were studied. The conventional transformation course featured four stages. Firstly, CMP and choline chloride were phosphorylated and CDP-choline was formed rapidly; secondly, the rate of CDP-choline formation declined and CMP was not detected in the mixture; thirdly, CMP was released and the CDP-choline concentration reached a peak; Fourthly, the compound concentrations did not practically changes eventually. Using the central composite design, the concentration, yield, and utilization efficiency of energy reached 24.7 mmol/L, 82.3% and 10.6%, with 30 mmol/L of ammonium ion and 1% (V/V) of glycerol, respectively. Ammonium ion not only strengthened the glycolytic pathway, but also coordinated the reaction rate between the glycolytic pathway and the Kennedy pathway. Glycerol alleviated the activity decrease of the key enzymes in the mixture.

A new member of the short-chain dehydrogenases/reductases superfamily: purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis.

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS-PAGE and only required NADPH as an electron donor. The K(m) value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V(max) was 337 micromol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or alpha,beta-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and beta-mercaptoethanol, but not by ethylene diamine tetra acetic acid.