Construction of an artificial consortium of Escherichia coli and cyanobacteria for clean indirect production of volatile platform hydrocarbons from CO2.

Ethylene and isoprene are essential platform chemicals necessary to produce polymers and materials. However, their current production methods based on fossil fuels are not very efficient and result in significant environmental pollution. For a successful transition more sustainable economic model, producing these key polymeric building blocks from renewable and sustainable resources such as biomass or CO2 is essential. Here, inspired by the symbiotic relationship of natural microbial communities, artificial consortia composed of E. coli strains producing volatile platform chemicals: ethylene and isoprene and two strains of cyanobacteria phototrophically synthesizing and exporting sucrose to feed these heterotrophs were developed. Disaccharide produced by transgenic cyanobacteria was used as a carbon and electron shuttle between the two community components. The E. coli cscB gene responsible for sucrose transport was inserted into two cyanobacterial strains, Thermosynechococcus elongatus PKUAC-SCTE542 and Synechococcus elongatus PCC7942, resulting in a maximal sucrose yield of 0.14 and 0.07 g/L, respectively. These organisms were co-cultured with E. coli BL21 expressing ethylene-forming enzyme or isoprene synthase and successfully synthesized volatile hydrocarbons. Productivity parameters of these co-cultures were higher than respective transgenic cultures of E. coli grown individually at similar sucrose concentrations, highlighting the positive impact of the artificial consortia on the production of these platform chemicals.

Discovery of Five New Ethylene-Forming Enzymes for Clean Production of Ethylene in E. coli.

Ethylene is an essential platform chemical with a conjugated double bond, which can produce many secondary chemical products through copolymerisation. At present, ethylene production is mainly from petroleum fractionation and cracking, which are unsustainable in the long term, and harmful to our environment. Therefore, a hot research field is seeking a cleaner method for ethylene production. Based on the model ethylene-forming enzyme (Efe) AAD16440.1 (6vp4.1.A) from Pseudomonas syringae pv. phaseolicol, we evaluated five putative Efe protein sequences using the data derived from phylogenetic analyses and the conservation of their catalytic structures. Then, pBAD expression frameworks were constructed, and relevant enzymes were expressed in E. coli BL21. Finally, enzymatic activity in vitro and in vivo was detected to demonstrate their catalytic activity. Our results show that the activity in vitro measured by the conversion of α-ketoglutarate was from 0.21-0.72 µmol ethylene/mg/min, which varied across the temperatures. In cells, the activity of the new Efes was 12.28-147.43 µmol/gDCW/h (DCW, dry cellular weight). Both results prove that all the five putative Efes could produce ethylene.

[Down-regulation of tripartite motif containing 59 (TRIM59) blocks the NF-κB signaling pathway and inhibits the invasion and migration of nasopharyngeal carcinoma cells].

Objective To investigate whether tripartite motif containing 59 (TRIM59) influences the biological behavior of nasopharyngeal carcinoma cells by regulating the nuclear factor κB(NF-κB) signaling pathway. Methods TCGA database was used to predict the expression of TRIM59 in nasopharyngeal carcinoma and adjacent tissues. Reverse transcription PCR and Western blot were used to detect the relative expressions of TRIM59 mRNA and protein in nasopharyngeal carcinoma and paracancerous tissues. With human normal nasal mucosal epithelial cells (HNEpCs) as a control, reverse transcription PCR and Western blot analysis were used to detect the relative expression of TRIM59 mRNA and protein in HNE1 and CNE-2Z nasopharyngeal carcinoma cells. Small interference RNA technology was used to down-regulate the level of TRIM59 in HNE1 cells, while a control group, small interference RNA negative control (si-NC) group and TRIM59 small interference RNA (si-TRIM59) group were set up. MTT assay was used to detect the cell proliferation inhibition rate of each transfection group. TranswellTM chamber was used to detect cell invasion and migration ability of each transfection group. Western blot analysis was employed to detect NF-κB and phosphorylated NF-κB (p-NF-κB), vimentin, survivin protein expression. ELISA was adopted to detect interleukin 6 (IL-6), IL-8, tumor necrosis factor α (TNF-α) in the supernatant of cultured cells. Results The expression of TRIM59 increased abnormally in nasopharyngeal carcinoma tissues. Compared with HNEpC cells, the relative expression of TRIM59 in HNE1 significantly increased. After down-regulating the expression of TRIM59 in HNE1 cells, cell survival and cell invasion and migration were significantly inhibited. Down-regulating the expression of TRIM59 inhibited HNE1 cells p-NF-κB, vimentin, survivin protein expression and significantly reduced the levels of IL-6, IL-8, and TNF-α. Conclusion Down-regulation of TRIM59 blocks the NF-κB signaling pathway and tumor inflammation-related factors in HNE1 cells, thereby inhibiting cell invasion and migration.

Development of silica molecularly imprinted polymer on carbon dots as a fluorescence probe for selective and sensitive determination of cetirizine in saliva and urine.

A fluorescence probe based on carbon dots (CDs) coated with silica molecularly imprinted polymer (MIPs) was synthesized for selective and sensitive determination of cetirizine (CTZ). Green source carbon dots were firstly derived from orange peels through a microwave method, and had the merits of eco-friendly and low toxicity. Then a thin silica film was formed on the surface of CDs by reverse microemulsion technique, and molecularly imprinted polymer coated on silica-carbon dots. In this scene, CTZ, 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS) were employed as a template, a functional monomer and cross linker, respectively. The obtained CDs-MIPs can selectively bind CTZ through the specific interaction between recognition sites and template, and obey photoinduced electron transfer fluorescence quenching mechanism. Fluorescence dropped linearly in the range of 0.5-500 ng mL-1, under the optimal conditions, with a detection limit of 0.41 ng mL-1. Furthermore, the proposed method was successfully intended for the determination of trace CTZ in human saliva and urine samples without the interference of other molecules and ions. And recoveries ranged from 95.8% to 99.8% with relative standard deviation less than 3.0%.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

The Genome Copy Number of the Thermophilic Cyanobacterium Thermosynechococcus elongatus E542 Is Controlled by Growth Phase and Nutrient Availability.

The recently isolated thermophilic cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542 (here Thermosynechococcus E542) is a promising strain for fundamental and applied research. Here, we used several improved ploidy estimation approaches, which include quantitative PCR (qPCR), spectrofluorometry, and flow cytometry, to precisely determine the ploidy level in Thermosynechococcus E542 across different growth stages and nutritional and stress conditions. The distribution of genome copies per cell among the populations of Thermosynechococcus E542 was also analyzed. The strain tends to maintain 3 or 4 genome copies per cell in lag phase, early growth phase, or stationary phase under standard conditions. Increased ploidy (5.5 ± 0.3) was observed in exponential phase; hence, the ploidy level is growth phase regulated. Nearly no monoploid cells were detected in all growth phases, and prolonged stationary phase could not yield ploidy levels lower than 3 under standard conditions. During the late growth phase, a significantly higher ploidy level was observed in the presence of bicarbonate (7.6 ± 0.7) and high phosphate (6.9 ± 0.2) at the expense of reduced percentages of di- and triploid cells. Meanwhile, the reduction in phosphates decreased the average ploidy level by increasing the percentages of mono- and diploid cells. In contrast, temperature and antibiotic stresses reduced the percentages of mono-, di-, and triploid cells yet maintained average ploidy. The results indicate a possible causality between growth rate, stress, and genome copy number across the conditions tested, but the exact mechanism is yet to be elucidated. Furthermore, the spectrofluorometric approach presented here is a quick and straightforward ploidy estimation method with reasonable accuracy.IMPORTANCE The present study revealed that the genome copy number (ploidy) status in the thermophilic cyanobacterium Thermosynechococcus E542 is regulated by growth phase and various environmental parameters to give us a window into understanding the role of polyploidy. An increased ploidy level is found to be associated with higher metabolic activity and increased vigor by acting as backup genetic information to compensate for damage to the other chromosomal copies. Several improved ploidy estimation approaches that may upgrade the ploidy estimation procedure for cyanobacteria in the future are presented in this work. Furthermore, the new spectrofluorometric method presented here is a rapid and straightforward method of ploidy estimation with reasonable accuracy compared to other laborious methods.

Development of a sample pretreatment device integrating ultrasonication, centrifugation and ultrafiltration, its application on rapid on-site screening of illegally added chemical components in heat-clearing, detoxicating Chinese patent medicines followed by electrospray ionization-ion mobility spectrometry.

In this paper, a simple and rapid sample pretreatment device integrating ultrasonication, centrifugation and ultrafiltration (UCU) was reported for preparation of trace analytes in complex matrices. The UCU device was composed of two parts, A and B. The sample and extraction solvent were put into Part B for ultrasonic extraction. Subsequently, Part A and Part B were integrated and sealed for centrifugation and ultrafiltration. Finally, the ultrafiltrate in Part A was taken out for subsequent detection. After optimization, the device was applied to rapid on-site screening of five illegally added chemical components in heat-clearing and detoxicating Chinese patent medicines by combining with electrospray ionization-ion mobility spectrometry (ESI-IMS). The method showed good performance in terms of linearity with correlation coefficients (R2) above 0.9976 and limits of detection (LODs) in the range of 0.049-0.391 µg mL-1. The recoveries were from 96.5 % to 100.8 %. The whole analysis process was within 11 min. The proposed method was further compared with other methods reported in the literature and the advantages and considerations were also explored. The results demonstrated that it was a simple, fast and accurate technique. The establishment of this method not only greatly improved the experimental efficiency but also avoided potential sample pollution brought by multiple sample transfer, and could provide a powerful means for rapid on-site analysis of trace analytes in complex matrices.

A fast and simple approach for the quantification of five anti-hypersensitivity drugs in saliva and urine by portable ion mobility spectrometry based on magnetic graphene oxide dispersive solid phase extraction.

Fatal road traffic crashes are often related to multifarious risk factors, among which driving under the influence of drugs (DUID) has been reported as a significantly contributing cause. The first worry about the side-effect of influencing driving drugs is central nervous system adverse reaction, and anti-hypersensitivity drugs are a class of drugs with such side effects meanwhile has been widely used for common allergic diseases thus posing a great challenge to road safety and demanding a rapid and efficient method to detect. In this work, a method based on magnetic graphene oxide dispersive solid phase extraction (MGO-D-SPE) combined with ion mobility spectrometry (IMS) was firstly introduced for simultaneous determination of ephedrine, pseudoephedrine, diphenhydramine, promethazine and terfenadine in saliva and urine matrices. The prepared MGO was characterized by Fourier transform infrared (FT-IR) spectroscopy and thermo gravimetric analysis (TGA). Various parameters affecting extraction efficiency as well as instrumental acquisition sensitivity were studied and optimized. Under the optimum experimental conditions, the method was fully validated and the results demonstrated that the proposed method exhibited some advantages, including a good linearity covering large concentration ranges of 51.0-3040 ng ml-1 for five anti-hypersensitivity drugs, and good accuracy was also obtained with high precision (CV% < 5.0 %). LODs and LOQs were 10.2-50.4 ng·mL-1 and 30.6-101.3 ng·mL-1, respectively. Consequently, the MGO-D-SPE-IMS methodology succeeded in building a hitherto unexplored tool for quantifying anti-hypersensitivity drugs in saliva and urine matrices of interest in DUID research field.

MTH1 Inhibitor TH287 Suppresses Gastric Cancer Development Through the Regulation of PI3K/AKT Signaling.

Background: Cancer cells evade oxidative stress through the MutT homologue-1 (MTH1), a member of the Nudix family. MTH1 maintains genome integrity and the viability of tumor cells. A new class of MTH1 inhibitors have attracted interest as anticancer agents, but their mechanisms of action remain poorly characterized. In this study, the authors evaluated the anticancer effects of the MTH1 inhibitor TH287 on gastric cancer (GCa) cells. Materials and Methods: BGC-823 and SGC-7901 cells were treated with TH287 and CCK-8, and colony-forming assays were performed. Cell migration was assessed through Transwell and scratch assays. Apoptotic status was measured via flow cytometry and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining. Cell cycle status was assessed by propidium iodide (PI) staining. The expression of PI3K/AKT signaling-related proteins was verified by western blotting. Results: TH287 inhibited cell viability, reduced cell proliferation, inhibited apoptosis, induced G2/M arrest, and suppressed cell migration. A loss of mitochondrial membrane potential and reduced Bcl-2/Bax expression were also observed in TH287-treated cells. These effects were mediated through the inhibition of pro-oncogenic PI3K/AKT signaling. Conclusions: These findings indicate that the MTH1 inhibitor TH287 mediates an array of anticancer effects in GCa cells through its effects on mitochondrial function and PI3K/AKT signaling. Collectively, these data highlight the promise of TH287 as a novel therapeutic option for GCa cells.

miR­146a improves hepatic lipid and glucose metabolism by targeting MED1.

Non­alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. Increasing evidence has shown that microRNAs (miRNAs) play a vital role in the progression of NAFLD. The aim of the present study was to examine the expression level and roles of miR­146a in fatty liver of high­fat diet (HFD) and ob/ob mice and fatty acid­treated hepatic cells using RT­qPCR and western blot analysis. The results showed that the expression of miR­146a was significantly decreased in the livers of high­fat diet (HFD) and ob/ob mice and free fatty acid­stimulated cells by RT­qPCR. Overexpression of hepatic miR­146a improved glucose and insulin tolerance as well as lipid accumulation in the liver by promoting the oxidative metabolism of fatty acids. In addition, the overexpression of miR­146a increased the amount of mitochondria and promoted mitochondrial respiration in hepatocytes. Similarly, inhibition of miR­146a expression levels significantly reduced mitochondrial numbers in AML12 cells as well as the expression of mitochondrial respiration related genes. Additionally, MED1 was a direct target of miR­146a and restoring MED1 abolished the metabolic effects of miR­146a on lipid metabolism and mitochondrial function. Therefore, results of the present study identified a novel function of miR­146a in glucose and lipid metabolism in targeting MED1, suggesting that miR­146a serves as a potential therapeutic target for metabolic syndrome disease.

Long non-coding RNA ANRIL enhances mitochondrial function of hepatocellular carcinoma by regulating the MiR-199a-5p/ARL2 axis.

Although the roles of long non-coding RNA (lncRNA) ANRIL (Antisense non-coding RNA in the INK4A locus) have been established in various tumors, its roles in mitochondrial metabolic reprogramming of hepatocellular carcinoma (HCC) cells are still unclear. This work aims to explore lncRNA ANRIL roles in regulating the mitochondrial metabolic reprogramming of liver cancer cells. First, we found that lncRAN ANRIL expression was significantly increased in HCC tissues or cells compared with the normal adjacent tissues and normal tissues or cells. Functional experiment showed that overexpression of lncRNA ANRIL promoted mitochondrial function in HCC cells, evident by the increased mitochondrial DNA copy numbers, ATP (Adenosine triphosphate) level, mitochondrial membrane potential, and the expression levels of mitochondrial markers, while ANRIL knockdown exerted the opposite effects. Mechanistically, lncRNA ANRIL acted as a competing endogenous RNA to increase ARL2 (ADP-ribosylationfactor-like 2) expression via sponging miR-199a-5p. Notably, the miR-199a-5p/ARL2 axis is necessary for ANRIL-mediated promoting effects on HCC cell mitochondrial function. This work reveals a novel ANRIL-miR-199a-5p-ARL2 axis in HCC cell progression, which might provide potential targets for HCC treatment.

Dispersive liquid-liquid microextraction with high-performance liquid chromatography for the analysis of 1,4-benzodioxane-6-aldehyde in eliglustat tartrate active pharmaceutical ingredient.

Potential genotoxic impurities (PGIs) are a series of compounds that could potentially damage DNA. Therefore, a sensitive method is needed for detection and quantification. The present work described and validated a method for the quantification of one PGI (namely 1,4-benzodioxane-6-aldehyde) in Eliglustat tartrate (EGT) active pharmaceutical ingredient (API) substances using dispersive liquid-liquid microextraction (DLLME) as sample preparation to remove matrix effect and detected by HPLC-UV. Parameters influencing the microextraction efficiency were systematically investigated. The combined application of DLLME and HPLC-UV provided the sensitivity of the method. The achieved limit of detection (LOD) and the limit of quantification (LOQ) were adequate for the specific purpose and found to be 1.29 µg g-1 and 2.58 µg g-1, respectively. This simple and effective methodology offers a key advantage in the ease of removing matrix effect and improves sensitivity obviously. In addition, no costly instrumentation and skilled personnel are needed when using this method, which is available and can be successfully implemented in routine factory drug quality control analysis.

Gastrodin Suppresses Pentylenetetrazole-Induced Seizures Progression by Modulating Oxidative Stress in Zebrafish.

Pentylenetetrazole (PTZ)-induced seizures in Zebrafish models are now widely accepted for investigating human disease epilepsy. In epilepsy, the generation of oxidative stress contributes to the brain injury. Although Gastrodin (GAS) has been reported to have anticonvulsant activities, its effects on zebrafish seizure models and the underlying mechanism remain unclear. In this study, we evaluated the effects of GAS pretreatment on PTZ-induced seizures in zebrafish larvae and investigated the underlying mechanism related to its anti-oxidative defense. We found for the first time that GAS significantly decreased seizure-like behavior and extended the latency period to the onset of seizures. In addition, after exposure to GAS, anti-oxidative activity was observed in PTZ-induced seizures by measurement of antioxidant enzymes activities and oxidative stress-related genes expression. The overall results indicate that GAS attenuates PTZ-induced seizures in a concentration-dependent manner and modulates oxidative stress to potentially protect larval zebrafish from further seizures. Furthermore, our results have provided novel insights into GAS related therapy of seizures and associated neurological disorders.