Triphosphate Content Research Articles

Dexamethasone enhances glucose uptake by SGLT1 and GLUT1 and boosts ATP generation through the PPP-TCA cycle in bovine neutrophils.

BackgroundClinical dexamethasone (DEX) treatment or stress in bovines results in extensive physiological changes with prominent hyperglycemia and neutrophils dysfunction.ObjectivesTo elucidate the effects of DEX treatment in vivo on cellular energy status and the underlying mechanism in circulating neutrophils.MethodsWe selected eight-month-old male bovines and injected DEX for 3 consecutive days (1 time/d). The levels of glucose, total protein (TP), total cholesterol (TC), and the proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in blood were examined, and we then detected glycogen and adenosine triphosphate (ATP) content, phosphofructosekinase-1 (PFK1) and glucose-6-phosphate dehydrogenase (G6PDH) activity, glucose transporter (GLUT)1, GLUT4, sodium/glucose cotransporter (SGLT)1 and citrate synthase (CS) protein expression and autophagy levels in circulating neutrophils.ResultsDEX injection markedly increased blood glucose, TP and TC levels, the Ca2+/P5+ ratio and the neutrophil/lymphocyte ratio and significantly decreased blood IL-1β, IL-6 and TNF-α levels. Particularly in neutrophils, DEX injection inhibited p65-NFκB activation and elevated glycogen and ATP contents and SGLT1, GLUT1 and GR expression while inhibiting PFK1 activity, enhancing G6PDH activity and CS expression and lowering cell autophagy levels.ConclusionsDEX induced neutrophils glucose uptake by enhancing SGLT1 and GLUT1 expression and the transformation of energy metabolism from glycolysis to pentose phosphate pathway (PPP)-tricarboxylic acid (TCA) cycle. This finding gives us a new perspective on deeper understanding of clinical anti-inflammatory effects of DEX on bovine.

Evaluation of the membrane damage mechanism of thymol against Bacillus cereus and its application in the preservation of skim milk

The goal of this study was to elucidate the mechanism by which thymol caused membrane damages in Bacillus cereus cells and the feasibility of its application in the preservation of skim milk. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution method. The membrane potentials, intracellular adenosine triphosphate (ATP) concentrations, and pH gradients across the membrane were characterized using fluorescence spectroscopy. The degree of membrane damage and membrane structural integrity were observed under an electron microscope. The quantity of B. cereus in skim milk at different temperatures and concentrations of thymol was calculated using the plate counting method. As a result, the MIC of thymol against B. cereus BNCC 103930 was 0.625 mg/mL. Thymol significantly induced B. cereus cell dysfunction, membrane potential hyperpolarization, pH gradient dissipation, the blockade of ATP synthesis, and intracellular ATP depletion. Confocal laser scanning microscopy (CLSM) and field emission gun scanning electron microscope (FEGSEM) assays revealed pronounced membrane damages of B. cereus after thymol administration. Of note, low thymol concentrations, together with low-temperature storage, have the potential to control B. cereus in skim milk. These results indicated that thymol could be used as a food additive preventing B. cereus food poisoning. • The antimicrobial mechanism of thymol against Bacillus cereus was described. • Cell dysfunction induced by thymol was characterized using fluorescent probes. • Thymol causes morphological damages in Bacillus cereus. • Thymol controls the growth of Bacillus cereus in skim milk stored at low temperature.

Effects of Flavonoid Glycoside on Mitochondrial Dysfunction and Inflammatory Oxidative Stress in Macular Degeneration

This study aimed to investigate the effects of flavonoid glycoside on mitochondrial dysfunction and inflammatory oxidative stress in macular degeneration. Sixty specific-pathogen-free male Bagg and Albino mice were randomly divided into control, model and flavonoid glycoside. Human retinal pigment epithelium cells ARPE-19 were cultured and treated with dimethyl sulfoxide, sodium iodate and flavonoid glycoside respectively. Fundus fluorescein angiography was used to detect the number of mouse retinal leakage; optical coherence tomography retinal thickness. A wave and B wave were detected by electroretinogram. Hematoxylin and eosin staining was used to observe the morphology of mouse retina; phalloidin, translocase of the outer mitochondrial membrane complex subunit 20 and cyclooxygenase III staining were performed on mouse retinal pigment epithelium cells by immunofluorescence. Adenosine triphosphate content in retinal pigment epithelium cells of mice in each group was detected. Compared with the control mice, the number of retinal leakage points in the model increased, the retinal thickness, A wave and B wave decreased, and the levels of Fas and FasL protein increased significantly. After treatment with flavonoid glycoside, the number of retinal leakage decreased the retinal thickness, A wave and B wave increased, and the levels of Fas and FasL protein decreased significantly. Flavonoid glycoside can significantly improve the mitochondrial dysfunction and inflammatory oxidative stress of retinal pigment epithelium cells and play a good role in protecting macular degeneration.

Aptazyme-induced cascade amplification integrated with a volumetric bar-chart chip for highly sensitive detection of aflatoxin B1 and adenosine triphosphate

A simple aptazyme-induced cascade signal amplification (denoted as ACSA) was integrated with a triple-channel volumetric bar-chart chip (TV-Chip) to visually quantitate aflatoxin B1 (AFB1) and adenosine triphosphate (ATP). Bifunctional aptazymes consisting of aptamers and G-quadruplex-forming sequences were modified on magnetic silica nanoparticles (MSNPs) to construct sensing probes. The recognition of aptamers and targets leads to the formation of hemin/G-quadruplex (hGQ) DNAzymes, which can mimic the horseradish peroxidase (HRP)-accelerated signal enhancement reaction, enabling the polymerization of dopamine and subsequent deposition of polydopamine (PDA) on the MSNP probes, thereby providing abundant anchor sites to covalently immobilize numerous 4-mercaptophenylboric acid (4-MPBA)-modified platinum nanoparticles (PtNPs) (MPt). As a result, this strategy possesses high sensitivity by introducing a large number of PtNPs into the TV-Chip. The detection limits of AFB1 and ATP with 0.075 pM and 0.818 pM, respectively, were easily achieved without any additional instruments. In addition, the detection results of AFB1-spiked food samples were in good agreement with the commercial AFB1 ELISA kit, which verified the accuracy and reliability of this method. The ACSA-based TV-Chip would show great promise for on-site and real-time visual quantitation of trace targets.

Describing the adsorption of sodium tripolyphosphate on kaolinite surfaces in a saline medium by molecular dynamics

• First principle calculations parametrize triphosphate. • The effects of salt and additive concentration are relevant for optimal adsorption. • It is presented the main mechanism by which additives are adsorbed on kaolinite surfaces. • Hydrogen bond and cation bridge are the main interactions between surfaces and additives. • Adsorbed additives modify the kaolinite net surface charge. One of the challenging tasks in tailings management is dealing with clay-based minerals, which create constant problems, especially in pumping thickened slurries. For their treatment, a broad range of additives has been tested in situ, where sodium triphosphate has shown some successful preliminary advantages due to its ability to disperse particles. This work aims to evaluate the adsorption capacity of sodium triphosphate in kaolinite from a theoretical molecular approach that combines quantum calculations and molecular dynamics simulation. First, triphosphate was parameterized from quantum calculations (QM) to obtain the force field that describes the system using molecular dynamics (MD) simulation. Then, MD simulations described the behavior of triphosphate in saline solutions. The results indicated that triphosphate can self-aggregate and even more in the presence of NaCl salts. The adsorption of triphosphate on kaolinite surfaces was stable and formed mainly sodium-mediated bridge-type interactions between the charged groups. The adsorption was favorable when the triphosphate concentration increased, but in the presence of NaCl, it increased only up to concentrations of 0.06 M. These latter results can be attributed to the tendency to form aggregate over the interaction with kaolinite.

Microplastics boost the accumulation of tetrabromobisphenol A in a commercial clam and elevate corresponding food safety risks

Marine bivalve molluscs are one of the primary seafood for consumers. Inhabiting terrigenous pollutant-convergent coastal areas and feeding through seawater filtration, edible bivalves are subjected to waterborne emerging pollutants such as microplastics (MPs) and tetrabromobisphenol A (TBBPA). Nevertheless, the potential risks of consuming MP-TBBPA contaminated seafood are still largely unknown. With that, accumulation of TBBPA with and without the presence of MPs in a commercial bivalve species, blood clam (Tegillarca granosa), was determined in the present study. Meanwhile, corresponding target hazard quotients (THQs) as well as margins of exposure (MoEs) were estimated to evaluate the potential health risks for clam consumers. Furthermore, the impacts of pollutants accumulation on the detoxification process and energy supply were analysed. The data obtained demonstrated that MPs aggravate the accumulation of TBBPA in clams, leading to elevated potential food safety risks (indicated by higher THQ values and lower MoE values) for consumers. In addition, the in vivo contents of CYP1A1 and UDP-glucuronosyltransferase, the enzymatic activity of glutathione-S-transferase, and the expression levels of five detoxification-related genes were all dramatically suppressed by MP-TBBPA. Furthermore, clams exposed to MP-TBBPA had significantly lower adenosine triphosphate contents and lower pyruvate kinase and phosphofructokinase activities. These results indicated that the aggravation of TBBPA accumulation may be due to the hence disruption of detoxification process and limited energy available for detoxification.

Antibacterial Mechanism of 3-Carene against the Meat Spoilage Bacterium Pseudomonas lundensis and Its Application in Pork during Refrigerated Storage.

Pseudomonas lundensis is the main bacterium responsible for meat spoilage and its control is of great significance. 3-Carene, a natural monoterpene, has been proved to possess antimicrobial activities. This study aimed to investigate the antibacterial activity and mechanism of 3-carene against the meat spoilage bacterium P. lundensis, and explore its application on pork. After 3-carene treatment, cellular structural changes were observed. Cell walls and membranes were destroyed, resulting in the leakage of alkaline phosphatase and cellular contents. The decreased activity of Ca2+-Mg2+-ATPase and Na+-K+-ATPase showed the imbalance of intracellular ions. Subsequently, adenosine triphosphate (ATP) content and oxidative respiratory metabolism characteristics indicated that 3-carene inhibited the metabolism of the tricarboxylic acid cycle in P. lundensis. The results of binding 3-carene with the vital proteins (MurA, OmpW, and AtpD) related to the formation of the cell wall, the composition of the cell membrane, and the synthesis of ATP further suggested that 3-carene possibly affected the normal function of those proteins. In addition, the growth of P. lundensis and increase in pH were inhibited in pork during the 5 days of cold storage after the samples were pre-treated with 3-carene. These results show the anti-P. lundensis activity and mechanism of 3-carene, and its potential use in meat preservation under refrigerated conditions.

Toxicity mechanism of acrolein on DNA damage and apoptosis in BEAS-2B cells: Insights from cell biology and molecular docking analyses

Acrolein is a hazardous air pollutant for humans and is responsible for many pulmonary diseases, but the underlying mechanisms have not been completely elucidated. This work is focused on the genotoxicity effects of human bronchial epithelial (BEAS-2B) cells induced by acrolein (20, 40, 80 μM). The molecular mechanism was investigated base on DNA damage and mitochondrial apoptosis pathways. The results showed that after exposure to acrolein, the cell viability, glutathione (GSH) of BEAS-2B cells were reduced. Reactive oxygen species (ROS) level significantly increased, accompanied by increased levels of DNA damage-related indicators 8-hydroxy-2 deoxyguanosine (8−OHdG), DNA content of comet tail (Tail DNA%), olive tail moment (OTM), and nucleus morphology. Cell arrested at the G2/M phase. Then, the DNA damage response (DDR) signaling pathway (Ataxia-telangiectasia-mutated (ATM) and Rad-3-related (ATR)/Chk1 and ATM/Chk2) and the consequent cell cycle checkpoints were activated. The expression of γ-H2AX was significantly increased, indicating that acrolein induced DNA double-strand breaks. Molecular docking assay showed that acrolein bound to DNA in a spontaneous process. Moreover, mitochondrial apoptosis pathway involved in apoptosis, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) content of BEAS-2B cells were significantly reduced, and the apoptosis rate was significantly increased. The protein expression of Bax/Bcl-2 and Cleaved Caspase-3 were increased, and JNK signaling pathway was activated. All the results indicated that acrolein induced DNA damage, activated DDR and mitochondrial apoptosis pathways, which might be the pivotal factors to mediate cytotoxicity in BEAS-2B cells.

Bridging mRNA and Polycation Using RNA Oligonucleotide Derivatives Improves the Robustness of Polyplex Micelles for Efficient mRNA Delivery.

Polyplex for messenger RNA (mRNA) delivery requires strong yet reversible association between mRNA and polycation for extracellular robustness and selective intracellular disintegration. Herein, RNA oligonucleotide (OligoRNA) derivatives that bridge mRNA and polycation are developed to stabilize polyplex micelles (PMs). A set of the OligoRNAs introduced with a polyol moiety in their 5' end is designed to hybridize to fixed positions along mRNA strand. After PM preparation from the hybridized mRNA and poly(ethylene glycol)-polycation block copolymer derived with phenylboronic acid (PBA) moieties in its cationic segment, PBA moieties form reversible phenylboronate ester linkages with a polyol moiety at 5' end of OligoRNAs and a diol moiety at their 3' end ribose, in the PM core. The OligoRNAs work as a node to bridge ionically complexed mRNA and polycation, thereby improving PM stability against polyion exchange reaction and ribonuclease attack in extracellular environment. After cellular uptake, intracellular high concentration of adenosine triphosphate triggers the cleavage of phenylboronate ester linkages, resulting in mRNA release from PM. Ultimately, the PM provides efficient mRNA introduction in cultured cells and mouse lungs after intratracheal administration, demonstrating the potential of the bridging strategy in polyplex-based mRNA delivery.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO–) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO– and ATP. ONOO– selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO– and ATP products, ATP-LW allows ONOO– levels to be monitored in the green channel (λex = 488 nm, λem = 500–575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575–650 nm). The use of ATP-LW as a combined ONOO– and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO– donor).

Seawater desalination based drinking water: Microbial characterization during distribution with and without residual chlorine

Monitoring the changes that occur to water during distribution is vital to ensure water safety. In this study, the biological stability of reverse osmosis (RO) produced drinking water, characterized by low cell concentration and low assimilable organic carbon, in combination with chlorine disinfection was investigated. Water quality at several locations throughout the existing distribution network was monitored to investigate whether microbial water quality changes can be identified. Results revealed that the water leaving the plant had an average bacterial cell concentration of 103 cells/mL. A 0.5–1.5 log increase in bacterial cell concentration was observed at locations in the network. The residual disinfectant was largely dissipated in the network from 0.5 mg/L at the treatment plant to less than 0.1 mg/L in the network locations. The simulative study involving miniature distribution networks, mimicking the dynamics of a distribution network, fed with the RO produced chlorinated and non-chlorinated drinking water revealed that distributing RO produced water without residual disinfection, especially at high water temperatures (25–30 °C), poses a higher chance for water quality change. Within six months of operation of the miniature network fed with unchlorinated RO produced water, the adenosine triphosphate (ATP) and total cell concentration (TCC) in the pipe biofilm were 4 × 102 pg ATP/cm2 and 1 × 107 cells/ cm2. The low bacterial cell concentration and organic carbon concentration in the RO-produced water did not prevent biofilm development inside the network with and without residual chlorine. The bacterial community analysis using 16S ribosomal RNA (rRNA) gene sequencing revealed that mesophilic bacteria with higher temperature tolerance and bacteria associated with oligotrophic, nutrient-poor conditions dominated the biofilm, with no indication of the existence of opportunistic pathogenic species. However, chlorination selected against most bacterial groups and the bacterial community that remained was mainly the bacteria capable of surviving disinfection regimes. Biofilms that developed in the presence of chlorine contained species classified as opportunistic pathogens. These biofilms have an impact on shaping the water quality received at the consumer tap. The presence of these bacteria on its own is not a health risk indicator; viability assessment and qPCRs targeting genes specific to the opportunistic pathogens as well as quantitative microbiological risk assessment (QMRA) should be included to assess the risk.The results from this study highlight the importance of implementing multiple barriers to ensure water safety. Changes in water quality detected even when high-quality disinfected RO-produced water is distributed highlight microbiological challenges that chlorinated systems endure, especially at high water temperatures.

The Cell Protective Effect of Adenine on Hypoxia-Reoxygenation Injury through PPAR Delta Activation.

Ischemia followed by blood supply reperfusion in cardiomyocytes leads to an overproduction of free radicals and a rapid decrease of adenosine triphosphate concentration. The cardioprotective effect of a potential drug, adenine, was evaluated using H9c2 rat cardiomyoblasts. After hypoxia–reoxygenation (HR) treatment consisting of hypoxia for 21 h followed by reoxygenation for 6 h, it was revealed that pretreatment with 200 µM adenine for 2 h effectively prevented HR-induced cell death. Adenine also significantly decreased the production of reactive oxygen species and reduced cell apoptosis after HR injury. The antioxidant effect of adenine was also revealed in this study. Adenine pretreatment significantly reduced the expression of activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78) proteins, and protein disulfide isomerase induced a protective effect on mitochondria after HR stimulation. Intracellular adenosine monophosphate-activated protein kinase, peroxisome proliferator-activated receptor delta (PPARδ), and perilipin levels were increased by adenine after HR stimulation. Adenine had a protective effect in HR-damaged H9c2 cells. It may be used in multiple preventive medicines in the future.

Dietary Arginine Supplementation Improves Intestinal Mitochondrial Functions in Low-Birth-Weight Piglets but Not in Normal-Birth-Weight Piglets.

Our previous studies revealed that L-arginine supplementation had beneficial effects on intestinal barrier functions of low-birth-weight (LBW) piglets, which were associated with the enhanced antioxidant capacity. Moreover, mitochondrial functions are closely related to the redox state. This study was to explore potential mechanisms of L-arginine-induced beneficial effects against intestinal dysfunction by regulating mitochondrial function of LBW piglets. Twenty 4-day-old normal birth weight (NBW) piglets (BW: 2.08 ± 0.09 kg) and 20 LBW siblings (BW: 1.16 ± 0.07 kg) were artificially fed either a basal diet or a basal diet supplemented with 1.0% L-arginine for 21 d, respectively. Growth performance, intestinal morphology, redox status, mitochondrial morphology, and mitochondrial functions were examined. Data were subjected to two-way analysis of variance. LBW piglets presented lower (p < 0.05) ADG, shorter (p < 0.05) intestinal villus height, lower (p < 0.05) jejunal adenosine triphosphate (ATP) content and higher (p < 0.05) concentrations of Ca2+ and 8-OH-dG in jejunal mitochondria, compared with NBW piglets. Supplementation with 1.0% L-arginine significantly increased (p < 0.05) ADG, the activities of CAT, SOD, and GPx, intestinal villus height and mRNA abundances of ZO-1 (2-fold) in the jejunum of LBW piglets, but not in NBW piglets. Furthermore, the concentrations of ATP and the transcription of COX IV, COX V genes were up-regulated (p < 0.05) and the concentration of Ca2+ and 8-OH-dG were decreased (p < 0.05) in arginine-treated LBW piglets. The results suggest that mitochondrial morphology is affected, and mitochondrial functions are impaired in the jejunum of LBW piglets. While supplementation with 1.0% L-arginine relieved intestinal dysfunction through enhancing antioxidant capacity and improving mitochondrial functions via repairing mitochondrial morphology, normalizing mitochondrial calcium, and increasing ATP concentration in the jejunum of LBW piglets. However, supplementation with L-arginine has no significant beneficial effects on intestinal health in NBW piglets.

Mechanisms of acrolein induces toxicity in human umbilical vein endothelial cells: Oxidative stress, DNA damage response, and apoptosis.

Acrolein is a ubiquitous environmental pollutant that produced by the incomplete combustion of cigarette smoke, forest fires, petroleum fuels, plastic materials, and cooking fumes. Inhalation is a common form of people exposure to acrolein, increasing evidence demonstrates that acrolein impairs the cardiovascular system by targeting vascular endothelial cells. However, the molecular mechanism of the cytotoxicity of acrolein exposure on vascular endothelial cells remains unclear. This work focused on the toxicity of acrolein on human umbilical vein endothelial cells (HUVECs). The molecular mechanism was studied based on oxidative stress, DNA damage response (DDR), and mitochondrial apoptosis pathways. After HUVECs were treated with 12.5, 25, and 50 μM acrolein for 24 h, cell viability, cell colony formation, mitochondrial membrane potential, and adenosine triphosphate content significantly reduced, and acrolein increased intracellular reactive oxygen species, apoptosis rate, and 8-hydroxy-2 deoxyguanosine (8-OHdG) level. Furthermore, p38MAPK and c-Jun N-terminal kinase signaling pathways were activated in response to oxidative stress. Moreover, acrolein induced G0/G1phase arrest, promoted the expression of γ-H2AX, activated the DDR signaling pathway (Ataxia-Telangiectasia-Mutated [ATM] and Rad-3-related/Chk1 and ATM/Chk2), and triggered the consequent cell cycle checkpoints. Finally, the protein expression of Bax/Bcl-2 and cleaved Caspase-3 was up-regulated, suggesting apoptosis was induced by triggering the mitochondrial apoptosis pathway. All these results indicated that acrolein induced HUVECs cytotoxicity by regulating oxidative stress, DNA damage, and apoptosis. This study provides a novel perspective on the mechanism of acrolein-induced cardiovascular toxicity, it will be helpful for the prevention of acrolein-induced cardiovascular disease.

The effect of high-penetrating emission on the growth, superoxide accumulation and ATP/ADP ratio in Saccharomyces cerevisiae

Many kinds of physical fields are able to affect biological activity, such as electromagnetic field, magnetic field, scalar wave, magnetic vector potential and so on. Some works investigated a high-penetrating emission (HPE) generated by a LED generator that can affect the conductivity of water and the growth of dry yeast. In this research, the biological effect of the HPE of LED generator on S. cerevisiae was studied. The results showed that this kind of HPE could promote the growth of S. cerevisiae. With the decrease of initial concentration and the extension of exposure time, the growth promoting effect of HPE increased significantly. When the initial cell concentration was 2 × 102 cell/ml and exposed for 24 h, the number of yeast cells increased by 19.5% compared with the control group. Further studies showed that adenosine triphosphate (ATP) content and the ratio of ATP and adenosine diphosphate (ADP, ATP/ADP ratio) of exposed cells were higher than those of unexposed cells, indicating that exposed cells had higher cell viability and energy status. In addition, the intracellular superoxide (O2−) content decreased significantly after the HPE exposed. In this study, the stainless steel box acts as a Faraday cage to shield the electromagnetic field, so the biological effect of the HPE is not produced by the electromagnetic field. Nevertheless, the mechanism of the HPE is still unknown and needs further research. This is the first report on the effect of HPE on mitochondrial parameters, contributing to further study on HPE and revealing potential for future medical applications.

Bacteriostatic effects of high-intensity ultrasonic treatment on Bacillus subtilis vegetative cells

The bacteriostatic effects of high-intensity ultrasonic treatment (HIU) on Bacillus subtilis vegetative cells were evaluated, and the related mechanisms were explored using quantitative proteomics. The bacteriostatic effect of HIU on B. subtilis was proportional to the ultrasound treatment time and power, and the number of cultivable B. subtilis cells was decreased by approximately one log (at 270 W for 15 min) or half log (at 90 W for 25 min or 360 W for 5 min). Scanning electron microscopy images and gel electrophoresis results showed that HIU caused the destruction of the cell structure and intracellular protein leakage. In addition, HIU treatment at 270 W for 15 min resulted in the greatest decrease (84.22%) in intracellular adenosine triphosphate (ATP) content. The quantitative proteomic analysis showed that B. subtilis resisted the stress of HIU treatment by regulating the key proteins in physiological activities related to membrane transport (ATP-binding cassette [ABC] transporter), signal transduction (the two-component system), and energy metabolism (the tricarboxylic acid [TCA] cycle). HIU-induced physical damage, stress, and metabolic disorders were the main causes of the bacteriostatic effects on B. subtilis. These findings provide a foundation for the subsequent optimization and potential applications of HIU inactivation of B. subtilis.

In vitro anticancer activity of eco-friendly synthesized ZnO/Ag nanocomposites

Ag nanoparticle-integrated ZnO nanoflakes (ZnO/Ag nanocomposite) were synthesized by a simple green method using Beta vulgaris (beetroot) extract and assessed for their potential in in vitro anticancer activity. Four Ag contents (2.5, 5, 7.5, and 10%) were used in the composite (ZnO/Ag-2.5, ZnO/Ag-5, ZnO/Ag-7.5, and ZnO/Ag-1.0, respectively) to examine the effects of Ag on the surface, structural, morphological, and bio-activities of the ZnO/Ag nanocomposite. Bandgap narrowing was observed due to the introduction of silver in the ZnO/Ag composites. These nanocomposites were examined for their cytotoxic effects in the cervical (HeLa) and ovarian (SKOV-3) carcinoma cell lines. The dose-dependent cytotoxicity was investigated by studying the cell viability assay, ROS generation, and adenosine triphosphate (ATP) contents when the cell lines were exposed to the ZnO/Ag composites for 48 h. Among the various ZnO/Ag composites produced, the ZnO/Ag 7.5 composite exhibited superior anticancer activity against HeLa and SKOV-3 cell lines. The Cell viability and ATP contents were reduced significantly in a dose-dependent manner, whereas ROS generation was increased appreciably.

Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPsand ADP in biological samples.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Examination of Extracellular Polymer (EPS) Extraction Methods for Anaerobic Membrane Bioreactor (AnMBR) Biomass

Membrane bioreactor fouling is a complex process, which is typically driven by extracellular polymeric substances (EPS), a complex mixture of polysaccharides, proteins, lipids, humic substances, and other intercellular polymers. While much is known about fouling in aerobic membrane reactors, far less is known about fouling in anaerobic membrane bioreactors (AnMBR). Much of this knowledge, including EPS extraction methods, has been extrapolated from aerobic processes and is commonly assumed to be comparable. Therefore, several extraction methods commonly used for aerobic EPS quantification, including ultrasonication, ethylenediaminetetraacetic acid (EDTA), and formaldehyde plus sodium hydroxide (CH2O+NaOH), were evaluated to determine the most suitable extraction method for EPS of anaerobic microorganisms in an AnMBR. To maximize EPS yields, each extraction was performed four times. Experimental results showed that the EDTA method was best for EPS quantification, based on chemical oxygen demand (COD), dissolved organic carbon (DOC), and protein yields: 1.43 mg COD/mg volatile suspended solids (VSS), 0.14 mg DOC/mg VSS, and 0.11 mg proteins/mg VSS. In comparison, the CH2O+NaOH method maximized the extraction of carbohydrates (0.12 mg carbohydrates/mg VSS). However, multiple extraction cycles with EDTA and ultrasonication exhibited lower extracellular adenosine triphosphate (ATP) concentrations compared to CH2O+NaOH extractions, indicating lower levels of released intracellular substances. Successive EPS extractions over four cycles are better able to quantify EPS from anaerobic microorganisms, since a single extraction may not accurately reflect the true levels of EPS contents in AnMBRs, and possibly in other anaerobic processes.

Effect and mechanism of circular RNA BICD2 on the biological behavior of oral squamous cell carcinoma cells

Objective: To investigate the effects of circular RNA (circRNA) BICD2 (circ-BICD2) on glutamine metabolism, cell proliferation, migration, invasion and apoptosis of oral squamous cell carcinoma (OSCC) cells and to further explore the possible mechanism. Methods: OSCC cells were purchased from Shanghai Institute of Cellular Cells, Chinese Academy of Sciences. Samples of OSCC tissues and corresponding adjacent tissues were collected from 35 OSCC patients who underwent surgical resection at the First Affiliated Hospital of Zhengzhou University from January 2016 to January 2018. Real-time quantitative PCR (RT-qPCR) and western blot were performed to detect the expression levels of circ-BICD2, miR-296-5p and transgelin2 (TAGLN2) in OSCC tissues and cells. Bioinformatics, dual luciferase report experiment and RNA immunoprecipitation (RIP) experiment were used to determine the targeting relationship between circ-BICD2 and miR-296-5p, miR-296-5p and TAGLN2. According to different transfection oligonucleotides or plasmids, CAL27 and SCC9 cells were divided into si-NC group (transfection si-NC), si-circ-BICD2 group (transfection si-circ-BICD2), si-circ-BICD2+anti-miR-NC group (transfected with si-circ-BICD2 and anti-miR-NC), si-circ-BICD2+anti-miR-296-5p group (transfected with si-circ-BICD2 and anti-miR-296-5p), miR-NC group (transfected with miR-NC), miR-296-5p group (transfected with miR-296-5p mimic), miR-296-5p+pcDNA (transfected with miR-296-5p mimic and pcDNA) and miR-296-5p+TAGLN2 group (transfected with miR-296-5p mimic and pcDNA-TAGLN2). Cell counting kit 8(CCK-8), colony formation experiment, flow cytometry, Scratch healing test and Transwell experiment were applied to detect OSCC cell viability, number of colonies, cycle distribution, apoptosis rate, migration rate and invasive cell numbers. Glutamine (gln) consumption, α-ketoglutaric acid (α-KG) production and adenosine triphosphate (ATP) concentration were also detected by the kit. The expression of cyclinD1 and Glutamine hydrolase (GLS1) proteins of CAL27 and SCC9 cells in each of the groups were detected by western blot. Twelve four-week-old clean BALB/c female nude mice were injected with a single-cell suspension of SCC9 cells into the axillary skin to establish a transplanted tumor model. Twelve transplanted tumor model mice were divided into sh-circ-BICD2 group and sh-NC group. Mice were sacrificed by cervical dislocation at 32 days after injection, the tumors were removed and the tumor weight was tested. Results: The expressions of circ-BICD2 (2.54±0.74) and TAGLN2 (1.86±0.15) were increased (P<0.05), while the expression of MiR-296-5p was decreased in OSCC tissues (P<0.05). Cell viability, clone formation numbers, migration rate, invasive cell numbers, S phase cell ratio, glu consumption, α-KG production, ATP concentration, expression of cyclinD1 and GLS1 proteins of OSCC cells were significantly reduced after interference with circ-BICD2 expression. The apoptosis rate, the proportion of cells in G0-G1 phase and the expression of miR-296-5p were significantly increased (P<0.05). Inhibiting the expression of miR-296-5p coulld reverse the effect of interfering with circ-BICD2 on OSCC cell proliferation, migration, invasion, apoptosis and glutamine metabolism (P<0.05). After overexpression of miR-296-5p, cell viability, clone formation red number, migration rate, number of invasive cells, S phase cell ratio, glu consumption, α-KG production, ATP concentration and expressions of cyclinD1, GLS1 and TAGLN2 proteins in OSCC cells were significant decrease. The rate of apoptosis and the proportion of cells in G0-G1 phase were significantly increased (P<0.05). Compared with overexpression of miR-296-5p, cell viability, clone formation red number, migration rate, number of invasive cells, S phase cell ratio, glu consumption, α-KG production, ATP concentration, cyclinD1, GLS1 and TAGLN2 proteins in OSCC cells after overexpression of miR-296-5p and TAGLN2 were significantly increased, and the apoptosis rate and the proportion of cells in the G0-G1 phase were significantly decreased (P<0.05). Compared with the sh-NC group, the tumor weights of mice in the sh-circ-BICD2 group were significantly reduced (P<0.05). Conclusions: Circ-BICD2 was highly expressed in OSCC cells. Interfering with circ-BICD2 could inhibit the proliferation, migration and invasion of OSCC cells, glutamine metabolism and tumor growth and promote cell apoptosis, which might be relate to the regulation of miR-296-5p/TAGLN2 molecular axis.