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Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

Through flow cytometry and qPCR technology, the promoter vector for high expression of eGFP protein in HEK293F cells was optimized and verified with hepatitis B surface antigen. The CAG promoter vector can not only effectively increase and maintain the expression level of recombinant proteins, but also increase the expression of transgenes under low temperature conditions., The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human‐like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT‐PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate‐early enhancer combined with the chicken beta‐actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta‐actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

There are many potential immunotherapeutic targets for cancer immunotherapy, which should be assessed for efficacy before they enter clinical trials. Here we established an

3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) are value-added chemicals with versatile applications in the chemical, pharmaceutical, and food industries. Nevertheless, sustainable production of 3-HP and 1,3-PDO is often limited by the lack of efficient strains and suitable fermentation configurations. Herein, attempts have been made to improve the co-production of both metabolites through metabolic engineering of Escherichia coli and process optimization. First, the 3-HP and 1,3-PDO co-biosynthetic pathways were recruited and optimized in E. coli, followed by coupling the pathways to the transhydrogenase-mediated cofactor regeneration systems that increased cofactor availability and product synthesis. Next, pathway rebalancing and block of by-product formation significantly improved 3-HP and 1,3-PDO net titer. Subsequently, glycerol flux toward 3-HP and 1,3-PDO synthesis was maximized by removing metabolic repression and fine-tuning the glycerol oxidation pathway. Lastly, the combined fermentation process optimization and two-stage pH-controlled fed-batch fermentation co-produced 140.50 g/L 3-HP and 1,3-PDO, with 0.85 mol/mol net yield.

BACKGROUND Hydrogen sulfide (H2 S) is a known signaling molecule in plants, which has the ability to delay fruit ripening. Our previous studies have shown that H2 S treatment could delay the maturation of kiwifruits by inhibiting ethylene production, improving protective enzyme activities, and decreasing the accumulation of reactive oxygen species to protect the cell membrane during storage. The mechanism related to the way in which H2 S affected kiwifruit maturation was still unclear. We performed transcriptome sequencing to explore the influences of H2 S on the softening of kiwifruit. RESULTS The firmness and the soluble solids content (SSC) of the kiwifruit were significantly better maintained with H2 S treatment compared to the control during the storage period (P

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Symmetrical bisamides of ethylene diamine of type ArCONHCH2CH2NHCOAr undergo anodic C–C bond cleavage in acetonitrile–LiClO4 under controlled-potential electrolysis. The electrogenerated carbocatio...

Antiviral drugs are among the most common and important classes of pharmaceuticals to treat viral infections, however their continuous emission and persistence in the receiving environment has attracted increasing attention about their potential ecological risks. Here we investigated the occurrence, fate and mass load of 9 antiviral drugs for acquired immunodeficiency syndrome and hepatitis B, in 7 wastewater treatment plants (WWTPs) with different treatment processes in Guangdong, China. Totally, 8 target antiviral drugs were detected in the WWTPs influent wastewater, effluent wastewater and sludge, with maximal concentrations up to 7624 ng/L (telbivudine), 568 ng/L (telbivudine), and 2013 ng/g wet weight (telbivudine), respectively. The removal efficiency varied widely between different antiviral drugs, with the mean aqueous removal efficiency and total removal efficiency ranging from -6.2% (nevirapine) to 100% (lamivudine) and -1.2% (nevirapine) to 100% (lamivudine), respectively. Mass balance analysis showed that their elimination was mostly attributed to the biodegradation/biotransformation. The total back-estimated usage and emission of 9 target antiviral drugs were 77.8 t/y and 13.2 t/y in Guangdong province, China, respectively. Based on the sewage epidemiology approach, the consumption and emission of antiviral drugs in seven studied WWTPs were ranged at 2.31 mg/d/1000 people (nevirapine) to 4970 mg/d/1000 people (telbivudine), and 0 (lamivudine) to 900 mg/d/1000 people (telbivudine), respectively. Preliminary risk assessment showed that the antiviral drugs of zidovudine, ritonavir, lopinavir, and telbivudine in the receiving rivers could pose high ecological risks for aquatic environment. The findings from the present study illustrate the persistence of nevirapine in WWTPs, and provide essential evidence for further study into the development of wastewater treatment technologies.

When dealing with the complex deformation of free surface such as wave breaking, traditional mesh-based Computational Fluid Dynamics (CFD) methods often face problems arising alongside grid distortion and re-meshing. Therefore, the meshless method became robust for treating large displaced free surface and other boundaries caused by moving structures. The particle method, which is an important branch of meshless method, is mainly divided into the Smoothed Particle Hydrodynamics (SPH) and Moving Particle Semi-implicit (MPS) methods. Different from the SPH method, which involves continuity and treat density as a variable when building kernel functions, the kernel function in the MPS method is a weight function which treats density as a constant, and the spatial derivatives are discretized by establishing the gradient operator and Laplace operator separately. In other words, the first- or second-order continuity of the kernel functions in the MPS method is not a necessity as in SPH, though it might be desirable. At present, the MPS method has been successfully applied to various violent-free surface flow problems in ocean engineering and diverse applications have been comprehensively demonstrated in a number of review papers. This work will focus on algorithm developments of the MPS method and to provide all perspectives in terms of numerical algorithms along with their pros and cons.

Background Low-dose amitriptyline (AMT) is an effective treatment for diarrhea-dominant irritable bowel syndrome (IBS-D). Its efficacy depends upon its serum concentration and the patient's CYP2C19 genotype. Aims To identify the association between serum AMT and nortriptyline (NT) concentration and CYP2C19 polymorphism and the clinical response in IBS-D patients. Methods Ninety IBS-D patients were treated of AMT for 6 weeks. Efficacy was evaluated by the results of the Adequate Relief question each week and an IBS severity scoring system (IBS-SSS) at 0, 3, and 6 weeks. CYP2C19 genotyping was performed by direct sequencing. AMT and NT steady-state serum concentrations were detected by high-performance liquid chromatography. Results The CYP2C19 polymorphism exhibited a significant influence on the NT serum concentration but did not predict the clinical efficacy of AMT for treating IBS-D. The NT steady-state and dose-corrected serum concentrations were significantly correlated with an improvement in the IBS-SSS score after 6 weeks, whereas the AMT serum concentration was not correlated with clinical improvement. The cut-off NT steady-state serum concentration of 2.91 ng/ml may help distinguish responders from non-responders. Conclusions NT serum concentration but not CYP2C19 polymorphism may be correlated with the clinical efficacy of AMT for treating IBS-D, and such a response may occur at the upper NT threshold of 2.91 ng/ml.

An electrochemical methodology for the Minisci-type trifluoromethylation of electron-deficient heterocycles mediated by cheap and easily available bromide ions has been developed. By virtue of the in situ generated sulfonyl hypobromite intermediate, the CF3 radical can be regulated and controlled at a low concentration, thereby improving the reaction efficiency over direct electrolysis. Also, this indirect electrochemical process is performed in a beaker-type undivided cell under galvanostatic conditions, without using external expensive supporting electrolytes. This protocol provides an alternative electrochemical trifluoromethylation methodology for the late-stage functionalization of biologically important molecules.

Trace antiviral drug contamination in aquatic ecosystems is becoming a significant environmental concern that requires an urgent efficient determination method. Here we developed sensitive and robust multi-residue determination methods to simultaneously extract and analyze 9 commonly used antiviral drugs (abacavir, zidovudine, efavirenz, nevirapine, ritonavir, lopinavir, lamivudine, telbivudine and entecavir) in surface water, wastewater, sediment, and sludge. Water samples were extracted with solid-phase extraction (SPE) technique using tandem hydrophilic–lipophilic balance and graphitized carbon black cartridges, while sediment and sludge samples were extracted using QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. The extraction conditions of SPE (pH and cartridge type) and QuEChERS (acetic acid content, salts reagent, and purification sorbent) methods were carefully optimized. We observed that under optimum conditions, the method quantification limits of the 9 antiviral drugs in water and solid samples ranged from 0.05 to 19.23 ng L−1 and from 0.02 to 7.38 ng g−1, respectively. For environmental samples spiking 3 different concentrations, the recovery values for the most targeted antiviral drugs ranged from 70 to 130%, except for efavirenz. All targeted antiviral drugs were detected in wastewater samples except for entecavir. We also found abacavir, efavirenz, ritonavir, lopinavir, and telbivudine in sediment and sludge samples. Notably, telbivudine was identified in all environmental matrices, with a high concentration of 127 ng L−1 and 222 ng g−1 in water and sediment samples, respectively.

A novel thiosemicarbazide modified adsorbent (PAN(MW)-TSC) based on polyacrylonitrile fiber was successfully synthesized under microwave irradiation, which was applied for the uptake of Cd(II) and Pb(II) from aqueous solution subsequently. Microwave irradiation method is a new approach to achieve the modification and it turns out that just a 30min process is enough for the anchoring of functional groups in the fiber matrix. The surface characterization was performed by fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) elemental analysis (EA) and thermogravimetric analysis (TGA), indicating that the modification was successfully accomplished. Batch adsorption experiments including equilibrium isotherms, kinetics and the effects of pH and temperature on the adsorption of Cd(II) and Pb(II) were systematically studied. Among three kinetic models, the pseudo-second-order kinetic model provides the best correlation for the process. The nonlinear resolution of the Langmuir isotherm equation has been found to show the closest fit to the equilibrium date. Thermodynamic parameters, involving △G, △H and △S were also calculated from graphical interpretation of the experimental data, which suggest that metal ions adsorption onto PAN(MW)-TSC fibers is spontaneous and exothermic. Regeneration of PAN(MW)-TSC fibers loaded with metal ions was efficiently done with 0.5M HNO3, by which the investigated adsorbent could be used reproductively for five times with a small decrease in sorption capacity. The feasible preparation of PAN(MW)-TSC fibers with high adsorption capacities opens a new perspective in the potential application for wastewater treatment.

Nitric oxide (NO) is a signal molecule that can regulate fruit physiology. Several studies have indicated that NO can inhibit the ripening of kiwifruit, but the mechanism underlying this process is unresolved. This study used transcriptome analysis to identify the essential genes related to NO regulation during kiwifruit softening. NO gas fumigation (15 μl L-1) significantly delayed kiwifruit softening. There were 736 differentially expressed genes (DEGs) between the NO treatment and the control. The expression levels of polygalacturonase (PG), pectate lyase (PL), β-galactosidase (β-GAL), pectinesterase (PE), and the beta-amylases-related genes decreased in response to the NO treatment, while those of four genes encoding cellulose synthase increased. The expression of genes related to ethylene biosynthesis and signal transduction also differed; the expression levels of 1-aminocyclopropane carboxylic acid oxidase (ACO), the ethylene receptors (ERS1, ETR2), and the ethylene-responsive transcription factors (ERF016, ERF7, ERF010, ERF062, ERF110, ERF037, ERF008, ERF113, ERF12, ERF095) were lower in the NO-treated kiwifruit. Expression of the calcium ion (Ca2+) signal-related genes (CNGC1, CPK1, CIPK2, CML31, CML48, ZIFL1) significantly differed and may be involved in the regulation of the NO softening response. These findings add to our understanding of the molecular mechanisms of the NO-delayed softening response in kiwifruit.

In this study, a facile and highly efficient approach, the microwave-assisted (MW-aid) method, was applied for the synthesis of amidoxime-functionalized fibrous adsorbent (PANMW-AO fibers), which exhibited enhanced adsorption capacities for Cr(VI) in aqueous solution. The preparation condition was optimized under four independent variables including: MW power, mass of NH2OH·HCl, time and bath ratio, based on the Box-Behnken design with response surface methodology (RSM). A period of 1 × 5 min was determined to be the optimum microwave (MW) ageing time for the synthesis of PANMW-AO, which is dramatically faster than using conventional heating methods. Effects of pH, contact time, initial concentration of Cr(VI) and temperature on adsorption were investigated systematically by bath adsorption experiments. Nonlinear solutions of a pseudo-second-order kinetic model and Langmuir isotherm model were found to provide the closest fit to the experimental data for the adsorption process of Cr(VI), which indicated that chemisorption is the controlling mechanism and monolayer adsorption is dominant. Thermodynamic parameters revealed the spontaneity of the adsorption process, and higher temperature favored adsorption. The formation of PANMW-AO and Cr(VI)-adsorbed PANMW-AO has been characterized by FTIR, SEM/EDX and XPS instrumentations, which demonstrate that Cr(VI) was adsorbed by the amidoxime group via a surface complexation mechanism. The adsorption of Cr(VI) ions was hardly affected by common coexisting ions such as SO42−, NO3− and Cl−. High desorption efficiency (>90%) of Cr(VI) was achieved using 0.1 M H2SO4 as effluent, by which the investigated adsorbent could be used repeatedly five times with a small decrease in sorption capacity. Rapidity of synthesis and low cost, coupled with highly efficient and rapid adsorption of Cr(VI) ions, make PANMW-AO fibers an attractive adsorbent in the potential application for wastewater treatment.

Mono- and bis-dialkylaminophenylbuta-1,3-dienyl boron-dipyrromethenes (BODIPYs) 1–12 were synthesized in 36%–42% yields by a Knoevenagel-type condensation. The absorption and emission maxima (λabs = 614–739 nm; λem = 655–776 nm in CHCl3) of 1–12 covered from the visible to the near-infrared region. Probe 1 was ratiometric Vis pH probes. Such probe was almost non-fluorescent. Upon the protonation of the tertiary amine function of 1, the strong fluorescence (Φf = 0.97) was released and the florescence intensity was dramatically increased by one thousand folds. The sharp isosbestic points were discovered at 590 nm, which was a ratiometric pH probe.

The long term evolution (LTE) based smart grid communication network is a promising candidate solution for the future power grid. In this paper, we discuss three channel estimation schemes for the LTE-based smart grids, including least square (LS), minimum mean square error (MMSE), and discrete Fourier transform (DFT) based channel estimators. In the simulations, we compare these three estimators, the results show that the DFT-based channel estimator can achieve the best tradeoff between the performance and the implementation complexity.