Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (ß-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. ß-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

The acceleration of aerobic sludge granulation by alternating organic loading rate: Performance and mechanism.

As a highly promising treatment technology for wastewater, long start-up time is one of the bottlenecks hindering the widespread application of aerobic granular sludge (AGS). This study focused on exploring the possibility of alternating organic loading rate (OLR) in promoting AGS granulation. Under alternating OLR (3.6-14.4 kgCOD/m3·d), AGS granulation was significantly accelerated. The mean granule size under alternating load reached 234.6 µm at 17 d, while under constant OLR (7.2 kgCOD/m3·d), the mean granule size was only 179.2 µm. Moreover, the granule size maintained continuous growth even when the alternating OLR was changed to constant OLR. Alternating load significantly increased the content of extracellular polymeric substances (EPS), especially proteins (PN) in tightly bound EPS (TB-EPS), which was likely the main reason for accelerating AGS granulation. Moreover, alternating load reduced the hydrophilicity of EPS and promoted the content of proteins secondary structures that favored aggregation in TB-EPS, which were also beneficial for granulation. Microbial community results showed that alternating load might promote the enrichment of EPS producing bacteria, such as Thauera, Brevundimonas and Shinella. Meanwhile, the content of enzymes that regulated amino acids metabolism also increased under alternating load, which might be related to the increase of PN in EPS. These results further demonstrated that alternating load promoted granulation through EPS.

Corrosion resistance and antibacterial activity of procyanidin B2 as a novel environment-friendly inhibitor for Q235 steel in 1 M HCl solution.

Flavonoids, alkaloids, glucosides and tannins with good corrosion inhibition are the main natural components in plants. In this work, procyanidin B2 (PCB2), a natural flavonoid, was firstly isolated from Uncaria laevigata. Corrosion inhibition, chemical reactivity and adsorption of PCB2 on Q235 carbon steel were described by experimental and theoretical studies. The inhibition performance of PCB2 as a green corrosion inhibitor was evaluated by electrochemical and gravimetric tests. The binding active sites and activities thereof on the steel surface were illustrated by quantum chemistry, and the equilibrium configuration was predicted by molecular dynamics simulation. PCB2 exhibits good corrosion inhibition on Q235 steel over a wide temperature range. The electrochemical results show that PCB2 is a mixed inhibitor, and its inhibition efficiency increases with the addition of PCB2 concentration. Moreover, the protective film is formed on the steel and the active corrosion sites are blocked significantly by surface analysis. Additionally, the theoretical calculation proves a strong interaction between PCB2 molecule and carbon steel. Besides, the antimicrobial activity was also preliminarily studied. This suggests that PCB2 exhibits better antimicrobial activity against many Gram-positive and Gram-negative bacteria. As a novel green corrosion inhibitor and antimicrobial agent, PCB2 is worthy of further exploitation.

Construction of an Escherichia coli cell factory to synthesize taxadien-5α-ol, the key precursor of anti-cancer drug paclitaxel.

Paclitaxel (Taxol™), an alkaloid of diterpenoid family, is one of the most widely used anti-cancer drugs due to its effectiveness against a variety of tumors. Rather than directly extraction and chemical synthesis of paclitaxel or its intermediates from yew plants, construction of a microbial cell factory for paclitaxel biosynthesis will be more efficient and sustainable. The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol. In this study, we report a recombinant Escherichia coli strain constructed with a heterologous mevalonate pathway, a taxadiene synthase from yew, and a cytochrome P450-mediated oxygenation system for the de novo production of taxadien-5α-ol, the first product of the multi-step taxadiene oxygenation metabolism. The key enzymes including taxadiene synthases and cytochrome P450 reductases were screened, and the linker for fusing taxadiene-5α-hydroxylase with its reductase partner cytochrome P450 reductase was optimized. By reducing the metabolic burden and optimizing the fermentation conditions, the final production of total oxygenated taxanes was raised up to 27 mg L-1 in a 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L-1, representing approximately a 12-fold and 23-fold improvements, respectively, as compared with the initial titers. The engineered MVA pathway for the overproduction of terpenoid precursors can serve as an efficient platform for the production of other valuable terpenoids.

[Curative effect of the combined therapy of warm acupuncture at back-shu points of five zang organs and western medicine on depression of yang deficiency pattern and its attenuating effect].

OBJECTIVE: To compare the therapeutic results and the occurrence of adverse reactions of medicine between the combined therapy of warm acupuncture at back-shu points of five zang organs and the western medicine and the simple wes-tern medication, and observe the therapeutic effect and attenuating effect of this combined therapy in treatment on depression of yang deficiency pattern. METHODS: A total of 80 patients with depression of yang deficiency pattern were randomly divided into an observation group and a control group （39 cases/group）. In the control group, escitalopram oxalate tablets were administered orally every day. In the observation group, on the base of the medication as the control group, warm acupuncture therapy was exerted at back-shu points of five zang organs, for 30 min each time, 5 times a week. The duration of treatment was 6 weeks in two groups. Before and after treatment, the scores of Hamilton depression scale (HAMD) and the antidepressant side effect scale (SERS) were evaluated and electroencephalogram (EEG) was detected; and the curative effect was assessed according to HAMD reduction rate in patients of the two groups. RESULTS: After treatment, the HAMD and SERS scores were lower than those before treatment in both groups. Compared with the control group, the HAMD and SERS scores were lower (P<0.05, P<0.01), and the EEG result was improved (P<0.01) in the observation group. The clinical total effective rate of the observation group was 97.43% (38/39), higher than 92.30% (36/39) of the control group (P<0.05). CONCLUSION: The combined therapy of warm acupuncture at back-shu points of five zang organs and western medicine effectively relieves depression of yang deficiency pattern in the patients and its overall therapeutic effect is better than simple western medication, besides, the combined therapy alleviates the adverse reactions induced by simple western medication.

The design, synthesis and anti-tumor mechanism study of new androgen receptor degrader.

Targeted protein degradation using small molecules is a novel strategy for drug development. In order to solve the problem of drug resistance in the treatment of prostate cancer, proteolysis-targeting chimeras (PROTAC) was introduced into the design of anti-prostate cancer derivatives. In this work, we synthesized two series of selective androgen receptor degraders (SARDs) containing the hydrophobic degrons with different linker, and then investigated the structure-activity relationships of these hybrid compounds. Most of the synthesized compounds exhibited moderate to good activity against all the cancer cell lines selected. Among them, compound A9 displayed potent inhibitory activity against LNCaP prostate cancer cell line with IC50 values of 1.75 µM, as well as excellent AR degradation activity. Primary mechanism studies elucidated compound A9 arrested cell cycle at G0/G1 phase and induced a mild apoptotic response in LNCaP cells. Further study indicated that the degradation of AR was mediated through proteasome-mediated process. For all these reasons, compound A9 held promising potential as anti-proliferative agent for the development of highly efficient SARDs for drug-resistance prostate cancer therapies.

Synthesis and in vitro biological evaluation of novel derivatives of Flexicaulin A condensation with amino acid trifluoroacetate.

As our research focus on anticancer drugs, two series of novel derivatives of Flexicaulin A (FA), an ent-kaurene diterpene, condensation with amino acid trifluoroacetate were synthesized, and their anti-proliferative activity against four human cancer cell lines (TE-1, MCF-7, A549 and MGC-803) were evaluated. Compared with FA, the anticancer activity and solubility of most derivatives were significantly improved. Among them, compound 6d had the best activity, and its IC50 value against Esophageal cancer cells (TE-1) was up to 0.75 µM. Subsequent cellular mechanism studies showed that compound 6d could inhibit the proliferation of cancer cells, the formation of cell colonies, and increase the level of ROS on TE-1 cells. In addition, 6d could up-regulate the expressions of SAPK/JNK pathway-associated proteins (p-ASK1, p-MKK4 and p-JNK) and pro-apoptotic proteins (Bak, Bad and Noxa), remarkably increase the ratio of Bax to Bcl-2 and activate Cleaved Caspase-3/9/PARP. These results indicate that compound 6d induces apoptosis through the ROS/JNK/Bcl-2 pathway and holds promising potential as an anti-proliferative agent.

Synthesis and In Vitro Antibacterial Activity of Quaternized 10-Methoxycanthin-6-one Derivatives.

BACKGROUND: Based on our previous work, we found that 10-methoxycanthin-6-one displayed potential antibacterial activity and quaternization was an available method for increasing the antibacterial activity. Here, we explored the antibacterial activity of quaternized 10-methoxy canthin-6-one derivatives. METHODS AND RESULTS: Twenty-two new 3-N-benzylated 10-methoxy canthin-6-ones were designed and synthesized through quaternization reaction. The in vitro antibacterial activity against three bacteria was evaluated by the double dilution method. Moreover, the structure-activity relationships (SARs) were carefully summarized in order to guide the development of antibacterial canthin-6-one agents. Two highly active compounds (6p and 6t) displayed 8-fold superiority (MIC = 3.91 µg/mL) against agricultural pathogenic bacteria R. solanacearum and P. syringae compared to agrochemical streptomycin sulfate, and showed potential activity against B. cereus. Moreover, these two compounds exhibited good "drug-like" properties, low cytotoxicity, and no inhibition on seed germination. CONCLUSIONS: This work provides two new effective quaternized canthin-6-one derivatives as candidate bactericide, promoting the development of natural-sourced bactericides and preservatives.

Synthesis and biological evaluation of novel Jiyuan Oridonin A-1,2,3-triazole-azole derivatives as antiproliferative agents.

As a continuation of our research on developing potent and potentially safe anti-proliferative agents, two series of novel Jiyuan Oridonin A-1,2,3-triazole-azole hybrids were designed, synthesized and evaluated for their anti-proliferative activity against four selected cancer cell lines (MGC-803, MCF-7, PC-3, Eca-109). Some compounds with better growth inhibitory effects were chosen to carry out further studies in A549 and SMMC-7721. Most of the synthesized compounds exhibited moderate to good activity against all the cancer cell lines selected. Particularly, the most active agent 8b showed high potency against human cancer cells with IC50 ranging from 0.2 ±â€¯0.0 to 5.0 ±â€¯0.9 µM. Cellular mechanism studies elucidated compound 8b arrests cell cycle at G1 phase and induce a strong apoptotic response in SMMC-7721 cells. Furthermore, 8b could inhibit the colony formation and migration via Wnt signaling pathway in SMMC-7721 cells. For all these reasons, compound 8b holds promising potential as anti-proliferative agent.

Clinical application of the fast track surgery model based on preoperative nutritional risk screening in patients with esophageal cancer.

OBJECTIVES: To investigate the clinical application of the fast track surgery (FTS) model based on preoperative nutritional risk screening (NRS) in patients with esophageal cancer. METHODS: 180 patients with esophageal cancer who underwent surgery between January 2008 and April 2014 were randomly divided into study and control groups based on matched-pairs. The study group underwent assessment using the NRS 2002 and received treatment before surgery and the control group was treated by the conventional method. Postoperative indicators including time to first exsufflation, time to defecation, time to chest tube removal, hospitalization duration, and postoperative complications were examined after surgery. RESULTS: Compared with the control group, the postoperative indicators including time to first exsufflation (88.4±2.76 vs 57.83±2.68 hours), time to first defecation (4.68±1.71 vs 3.28±1.34 days), time to chest tube removal (4.30±0.25 vs 2.70±0.33 days), postoperative hospitalization durations (11.71±1.39 vs 9.00±0.78 days), and total complication rate (18.9% [17/90] vs 6.67% [6/90]) were all significantly reduced in the study group (p<0.05). CONCLUSIONS: The FTS model based on NRS can effectively promote postoperative rehabilitation of patients, reduce the incidence of postoperative complications, and shorten hospital stay.

From HCOOH to CO at Pd electrodes: a surface-enhanced infrared spectroscopy study.

The decomposition of HCOOH on Pd surfaces over a potential range of practical relevance to hydrogen production and fuel cell anode operation was probed by combining high-sensitivity in situ surface-enhanced IR spectroscopy with attenuated total reflection and thin-layer flow cell configurations. For the first time, concrete spectral evidence of CO(ad) formation has been obtained, and a new main pathway from HCOOH to CO(ad) involving the reduction of the dehydrogenation product of HCOOH (i.e., CO(2)) is proposed.

Synthesis of a new chitosan derivative and assay of Escherichia coli adsorption.

A new chitosan derivative is prepared using chitosan. Ethyl cholorocarbonate was first introduced to the hydroxyl group of phthaloylchitosan through a nucleophilic reaction. Hydrazine was then added to recover the amino groups of chitosan, and promote cross-linking. The structure of this new chitosan derivative was characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and its physical properties were determined by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The thermal and chemical stabilities of the new derivative were improved compared with those of native chitosan. Assay of Escherichia coli adhesion on a film based on this chitosan derivative showed good adsorption and biofilm formation.

Microfluidics-based assay on the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors.

A new microfluidic system with four different microchambers (a circle and three equilateral concave polygons) was designed and fabricated using poly(dimethylsiloxane) (PDMS) and the soft lithography method. Using this microfluidic device at six flow rates (5, 10, 20, 30, 40, and 50 µL/h), the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors were investigated. Escherichia coli HB101 pGLO, which could produce a green fluorescent protein induced by l-arabinose, was utilized as the model bacteria. The results demonstrated that bacterial adhesion was significantly related to culture time, microenvironment geometry, and aqueous flow rates. Adhered bacterial density increased with the culture time. Initially, the adhesion occurred at the microchamber sides, and then the entire chamber was gradually covered with increased culture time. Adhesion densities in the side zones were larger than those in the center zones because of the lower shearing force in the side zone. Also, the adhesion densities in the complex chambers were larger than those in the simple chambers. At low flow rates, the orientation of adhered bacteria was random and disorderly. At high flow rates, bacterial orientation became close to the streamline and oriented toward the flow direction. All these results implied that bacterial adhesion tended to occur in complicated aqueous flow areas. The present study provided an on-chip flow system for physiological behavior of biological cells, as well as provided a strategic cue for the prevention of bacterial infection and biofilm formation.

Exploring electrosorption at iron electrode with in situ surface-enhanced infrared absorption spectroscopy.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) in attenuated total reflection (ATR) configuration has been extended to the Fe electrode/electrolyte interface in neutral and weakly acidic solutions for the first time. The SEIRA-active Fe film electrode was obtained through a potentiostatic electrodeposition of a virtually pinhole-free 40 nm-thick Fe overfilm onto a 60 nm-thick Au underfilm chemically predeposited on the reflecting plane of an ATR Si prism. The infrared absorption for CO adlayer at the Fe film electrode measured with ATR-SEIRAS was enhanced by a factor of larger than 34, as compared to that at a Fe bulk electrode with external infrared absorption spectroscopy in the literature. More importantly, the unipolar band shape enabled the reliable determination of the Stark tuning rates of CO adlayer at Fe electrode. In situ ATR-SEIRAS was also applied to study the electrosorption of a typical corrosion inhibitor benzotriazole (BTAH) on Fe electrode as a function of potential, providing additional spectral information at positive potentials in support of the formation of a polymer-like surface complex Fe(II)(m)(BTA)(n) as the corrosion-resistant layer.

Attenuated total reflection surface-enhanced infrared absorption spectroscopy at a cobalt electrode.

In situ surface-enhanced infrared absorption spectroscopy (SEIRAS) in attenuated total reflection (ATR) configuration has been extended to a Co electrode fabricated by potentiostatic deposition of a 50-nm-thick Co overlayer onto a Au underlayer chemically preformed on the reflecting plane of an ATR Si hemi-cylindrical prism. The as-prepared Co-on-Au film was characterized with atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The AFM images of the films before and after Co coating revealed island structures facilitating the SEIRA effect with Co nanoparticles much smaller than the underlying Au ones. The XPS spectrum did not contain any characteristic peaks related to Au, suggestive of a virtually pinhole-free nature of the Co overlayer. The voltammetric response of the as-prepared films in phosphate buffer solution (PBS, pH 6.9) was characteristic of a polycrystalline bulk Co electrode. Normally directed unipolar bands were found for surface probe CO molecules on Co surfaces in the PBS with their major band (CO(L)) intensity being one order of magnitude higher than that obtained with conventional IR reflection-absorption spectroscopy (IRRAS). By taking advantage of the higher detection sensitivity, the bands for linearly bonded CO (CO(L)) at 1965-2005 cm(-1) and the multi-bonded (CO(M)) band at 1845-1875 cm(-1) were clearly detected with their Stark tuning rates being 59 and 63 cm(-1) x V(-1), respectively, which would be otherwise unobtainable with the conventional IRRAS in the neutral solution.

Practically modified attenuated total reflection surface-enhanced IR absorption spectroscopy for high-quality frequency-extended detection of surface species at electrodes.

A practically modified ATR configuration has been proposed for in situ electrochemical surface-enhanced IR absorption spectroscopy (SEIRAS) by sandwiching an ultrathin water interlayer between a hemicylindrical ZnSe prism and a Si wafer as an integrated window. This new ATR optics significantly enhances the throughput of an effective IR beam across the ZnSe/gap/Si/metal film, enabling high-quality spectral fingerprints down to 700 cm(-1) to be readily detected at larger incidence angles without compromising the electrochemical feasibility and stability of metallic films deposited on Si. The advantages of this modified ATR-SEIRAS have been initially applied to explore two selected systems: wide-ranged in situ ATR-SEIRA spectra provided strong evidence in support of the formate intermediate pathway for methanol electrooxidation at the Pt electrode in an acid solution; in addition, new spectral fingerprints revealed comprehensive orientational information about of the p-nitrobenzoate species at Pt electrode as a result of the dissociative adsorption of p-nitrobenzoic acid molecules from an acid solution.

A microfluidic platform for sequential ligand labeling and cell binding analysis.

Developing biochemical and cell biological assay for screening biomolecules, evaluating their characteristics in biological processes, and determining their pharmacological effects represents a key technology in biomedical research. A PDMS-based integrated microfluidic platform was fabricated and tested for facilitating the labeling of ligand on the nanogram scale and sequential cell binding analysis in a manner that saves both time and reagents. Within this microfluidic platform, ligand labeling, cell immobolization, and optical analysis are performed in a miniaturized, continuous and semi-automated manner. This microfluidic device for ligand labeling and cell analysis is composed of two functional modules: (i) a circular reaction loop for fluorophore-labeling of the ligand and (ii) four parallel-oriented incubation chambers for immobilization of cells, binding of ligand to different cell populations, and optical evaluation of interactions between the labeled ligand and its cell targets. Epidermal growth factor (EGF) as the ligand and different cell lines with various levels of EGF receptor expression have been utilized to test the feasiblity of this microfluidic platform. When compared to studies with traditional Petri dish handling of cells and tissues, or even microwell analyses, experiments with the microfluidic platform described here are much less time consuming, conserve reagents, and are programmable, which makes these platforms a very promising new tool for biological studies.