ABSTRACT
Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a "fix and click" method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The "fix and click" strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without the production of sphingosine-1-phosphate. In contrast, when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without the formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using "fix and click" paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for the performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters, followed by the assay of enzyme activation at a later time and place.
Subject(s)
Lysophospholipids , Sphingolipids , Biological Assay , Ceramides/metabolism , Click Chemistry , Epithelial Cells/metabolism , Humans , Sphingolipids/metabolism , SphingosineABSTRACT
The type 2 diabetes mellitus (T2DM) is an urgent global health problem. T2DM patients are in a state of high oxidative stress and inflammation. Vitamin D and glutathione (GSH) play crucial roles in antioxidation and anti-inflammation. However, T2DM patients have lower vitamin D and GSH levels than healthy persons. A randomized controlled trial was conducted to see the effect of the vitamin D supplementation on oxidative stress and inflammatory factors in T2DM patients. In this study, a total of 178 T2DM patients were randomly enrolled, 92 patients received regular treatment (T2DM group) and 86 patients in Vitamin D group received extra vitamin D 400 IU per day in addition to regular treatment. Serum vitamin D, GSH, GSH metabolic enzyme GCLC and GR, inflammatory factor MCP-1, and IL-8 levels were investigated. We found that the T2DM group has significantly higher concentrations of MCP-1 and IL-8 than those in the healthy donor group. After vitamin D supplementation for 90 days, T2DM patients had a 2-fold increase of GSH levels, from 2.72 ± 0.84 to 5.76 ± 3.19 µmol/ml, the concentration of MCP-1 decreased from 51.11 ± 20.86 to 25.42 ± 13.06 pg/ml, and IL-8 also decreased from 38.21 ± 21.76 to 16.05 ± 8.99 pg/ml. In conclusion, our study demonstrated that vitamin D could regulate the production of GSH, thereby reducing the serum levels of MCP-1 and IL-8, alleviating oxidative stress and inflammation, providing evidence of the necessity and feasibility of adjuvant vitamin D treatment among patients with T2DM. On the other hand, vitamin D and GSH levels have important diagnostic and prognostic values in T2DM patients.
Subject(s)
Diabetes Mellitus, Type 2 , Vitamin D , Diabetes Mellitus, Type 2/drug therapy , Dietary Supplements , Glutathione , Humans , Inflammation , Interleukin-8/metabolism , Oxidative Stress , VitaminsABSTRACT
Spreading antimalarial resistance threatens effective treatment of malaria, an infectious disease caused by Plasmodium parasites. We identified a compound, BCH070, that inhibits asexual growth of multiple antimalarial-resistant strains of Plasmodium falciparum (half maximal inhibitory concentration [IC50] = 1-2 µM), suggesting that BCH070 acts via a novel mechanism of action. BCH070 preferentially kills early ring-form trophozoites, and, importantly, equally inhibits ring-stage survival of wild-type and artemisinin-resistant parasites harboring the PfKelch13:C580Y mutation. Metabolomic analysis demonstrates that BCH070 likely targets multiple pathways in the parasite. BCH070 is a promising lead compound for development of new antimalarial combination therapy that retains activity against artemisinin-resistant parasites.
Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Plasmodium falciparum/drug effects , Antimalarials/administration & dosage , Antimalarials/chemistry , Cells, Cultured , Drug Resistance , Fibroblasts/parasitology , Humans , Molecular Structure , Structure-Activity Relationship , Trypanosoma cruzi/drug effectsABSTRACT
The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.
Subject(s)
Cell Membrane/enzymology , Isoenzymes/chemistry , Isoenzymes/metabolism , Phospholipase C gamma/chemistry , Phospholipase C gamma/metabolism , Animals , Humans , Signal Transduction/physiologyABSTRACT
A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-ß3 by Gαq Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.
Subject(s)
Cell Membrane/enzymology , Fluorescence , Genes, Reporter , Phospholipase C beta/metabolism , Cell Membrane/genetics , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein beta Subunits/metabolism , HEK293 Cells , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphatidylinositol Phosphates/genetics , Phosphatidylinositol Phosphates/metabolism , Phospholipase C beta/geneticsABSTRACT
The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.
Subject(s)
Enzymes/metabolism , Fluorescent Dyes/chemistry , Phosphatidylinositols/metabolism , Hydrophobic and Hydrophilic Interactions , Lipids/chemistry , Liposomes/metabolismABSTRACT
All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-ß (PLC-ß) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-ß isozymes are autoinhibited, and several proteins, including Gαq, Gßγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gß1γ2 did not activate purified PLC-ß3 under these conditions despite their robust capacity to activate PLC-ß3 at membranes. In addition, mutants of PLC-ß3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-ß isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-ß isozymes.
Subject(s)
Cell Membrane/enzymology , Phospholipase C beta/metabolism , Allosteric Regulation , Animals , Biocatalysis , COS Cells , Chlorocebus aethiops , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Genes, Reporter , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Hydrolysis , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Models, Biological , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipase C beta/chemistry , Phospholipase C beta/isolation & purification , Protein Structure, Secondary , Protein Structure, Tertiary , SolubilityABSTRACT
Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.
Subject(s)
Enzyme Inhibitors/pharmacology , Type C Phospholipases/antagonists & inhibitors , Biological Assay/methods , Chemistry, Pharmaceutical/methods , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , HEK293 Cells , Humans , Isoenzymes , Models, Biological , Models, Chemical , Phospholipases/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Small Molecule Libraries , Substrate Specificity , Type C Phospholipases/metabolismABSTRACT
The extruded Mg-6Bi alloy and Mg-6Bi-1Ag alloy subjected to extrusion at 300 °C with the extrusion ratio of 25:1 and die-exit speed of 2 m/min were used to investigate microstructure characteristics and mechanical behavior. The experimental results demonstrate that the bimodal microstructure, composed of coarse dynamic unrecrystallized (unDRXed) grains and fine dynamic recrystallized (DRXed) grains, was obtained after extrusion. The Ag addition can obviously promote dynamic recrystallization and average grain size. It also indicates that the dynamic precipitation is significantly promoted by Ag addition during extrusion, obtaining more fraction of the Mg3Bi2 precipitates. Moreover, the extruded Mg-6Bi-1Ag alloy has a high tensile yield strength of 304 ± 2.0 MPa, which is increased by 19% compared to the extruded Mg-6Bi alloy, and elongation of 11.0 ± 1.7%, almost the same as 11.9 ± 0.9% of the extruded Mg-6Bi alloy. This result also shows that the extruded Mg-6Bi-1Ag alloy exhibits better strain hardening capacity. Therefore, Ag exhibits an effective role in promoting dynamic recrystallization and dynamic precipitation, resulting in the enhancement of strength and strain hardening capacity of the extruded Mg-6Bi-1Ag alloy, as well as keeping high ductility.
ABSTRACT
To investigate the influence of Zn-alloying on the microstructure and tensile mechanical properties of Mg-6Bi alloy after hot extrusion, a new ternary Mg-6Bi-3Zn alloy was prepared by extrusion at 300 °C. The microstructures, texture, dynamic precipitates and tensile mechanical behaviors of the extruded alloy were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), electron backscattered diffraction (EBSD) and a material testing machine at room temperature. After extrusion, the Mg-6Bi-3Zn alloy possesses a bimodal microstructure with elongated large unrecrystallized (unDRXed) grains and fine dynamic recrystallized (DRXed) grains. In addition, non-basal <202_1>//ED, <448_3>//ED and <112_1>//ED textures are observed within DRXed grains due to the Zn addition, leading to texture weakening in the extruded Mg-6Bi-3Zn alloy. Zn addition facilitates the dynamic precipitation behavior, leading to a 12.2% area fraction of Mg3Bi2 precipitates with an average size of 39.2 nm. Furthermore, incorporation of Zn atoms in Mg3Bi2 phases and segregation of Zn at the grain boundary are found. The extruded Mg-6Bi-3Zn alloy exhibits a tensile strength of 336 ± 7.1 MPa and a yield strength of 290 ± 5.5 MPa, as well as an elongation of 11.5%. Therefore, Zn addition is beneficial to enhance strength and keep good ductility for the extruded Mg-6Bi-3Zn alloy.
ABSTRACT
The aim of this work was to study the sublethal effects, biokinetics, subcellular partitioning and detoxification of arsenic in two native Chinses species, Bellamya quadrata and Cipangopaludina cathayensis, as well as an exotic South American species, Pomacea canaliculata. The exotic species exhibited higher tolerance than native species. Physiologically based pharmacokinetic model results showed that the exotic species P. canaliculata exhibited a lower bioaccumulation rate and a greater metabolism capacity of As. Subcellular partitioning of As revealed that P. canaliculata exhibits superior As tolerance compared to the native species B. quadrata and C. cathayensis. This is attributed to P. canaliculata effective management of the metal sensitive fraction and enhanced accumulation of As in the biologically detoxified metal fraction. Under As stress, the biochemical parameters (superoxide dismutase, malondialdehyde, glutathione and glutathione S-transferase) of the exotic species P. canaliculata changed less in the native species, and they returned to normal levels at the end of depuration period. Our study provides evidence of the superior survival capability of the exotic species P. canaliculata compared to the native species B. quadrata and C. cathayensis under environmentally relevant levels of As contamination.
Subject(s)
Arsenic , Snails , Animals , Snails/physiology , Arsenic/toxicity , Arsenic/metabolismABSTRACT
Naturally aged microplastics (NAMPs) and arsenic (As) have been reported to coexist in and threaten potentially to soil-plant ecosystem. The research explored the combined toxic effects of NAMPs and As to lettuce (Lactuca sativa L.) growth, and the distribution, accumulation and bioavailability of As in soil aggregates. The As contaminated soil with low, medium and high concentrations (L-As, M-As, H-As) were treated with or without NAMPs, and a total of six treatments. The results displayed that, in comparison to separate treatments of L-As and M-As, the presence of NAMPs increased the total biomass of lettuce grown at these two As concentrations by 68.9 % and 55.4 %, respectively. Simultaneous exposure of NAMPs and L-As or M-As led to a decrease in As content in shoot (0.45-2.17 mg kg-1) and root (5.68-14.66 mg kg-1) of lettuce, indicating an antagonistic effect between them. In contrast, co-exposure to H-As and NAMPs showed synergistic toxicity, and the leaf chlorophyll and nutritional quality of lettuce were also reduced. NAMPs altered the ratio of different soil aggregate fractions and the distribution of bioavailable As within them, which influenced the absorption of As by lettuce. In conclusion, these direct observations assist us in enhancing the comprehend of the As migration and enrichment characteristics in soil-plant system under the influence of NAMPs.
Subject(s)
Arsenic , Soil Pollutants , Arsenic/analysis , Lactuca , Microplastics , Plastics , Soil , Biological Availability , Ecosystem , Soil Pollutants/analysisABSTRACT
Francisella tularensis is a Gram-negative facultative intracellular bacterial pathogen that is classified by the Centers for Disease Control and Prevention as a Tier 1 Select Agent. F. tularensis infection causes the disease tularemia, also known as rabbit fever. Treatment of tularemia is limited to few effective antibiotics which are associated with high relapse rates, toxicity, and potential emergence of antibiotic-resistant strains. Consequently, new therapeutic options for tularemia are needed. Through screening a focused chemical library and subsequent structure-activity relationship studies, we have discovered a new and potent inhibitor of intracellular growth of Francisella tularensis, D8-03. Importantly, D8-03 effectively reduces bacterial burden in mice infected with F. tularensis. Preliminary mechanistic investigations suggest that D8-03 works through a potentially novel host-dependent mechanism and serves as a promising lead compound for further development.
Subject(s)
Anti-Bacterial Agents , Francisella tularensis , Tularemia , Francisella tularensis/drug effects , Francisella tularensis/growth & development , Animals , Tularemia/drug therapy , Tularemia/microbiology , Mice , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Structure-Activity Relationship , Humans , Microbial Sensitivity Tests , Drug Discovery , Female , Disease Models, AnimalABSTRACT
Proficiency testing based on quality control materials is an important component of the quality assurance system for detection methods. However, in the detection of infectious diseases, it is a challenge to use quality control materials derived from clinical samples or pathogens owing to their infectious nature. The Xpert MTB/RIF assay, endorsed by the World Health Organization, is one of the most widely implemented assays in the detection of Mycobacterium tuberculosis along with rifampicin resistance and its heterogeneity. Clinical isolates are typically used as quality controls for this assay, leading to concerns about biosafety, constrained target sequence polymorphisms, and time-consuming preparation. In this study, a heterogeneous quality control library for the Xpert MTB/RIF assay was constructed based on DNA synthesis and site-directed mutation, which provides sufficient rifampicin resistance polymorphisms, enabling monitoring all five probes of Xpert MTB/RIF and its combinations. Escherichia coli and Bacillus subtilis were used as heterogeneous hosts rather than the pathogen itself to eliminate biosafety risks; thus, preparation does not require a biosafety level III laboratory and the production time is reduced from a few months to a few days. The panel was stable for more than 15 months stored at 4°C and could be distributed at room temperature. All 11 laboratories in Shanghai participating in a pilot survey identified the specimens with corresponding probe patterns, and discordant results highlighted inappropriate operations in the process. Collectively, we show, for the first time, that this library, based on heterogeneous hosts, is an appropriate alternative for M. tuberculosis detection.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Rifampin/pharmacology , Drug Resistance, Bacterial/genetics , Sensitivity and Specificity , China , Tuberculosis/diagnosis , Tuberculosis/microbiology , Mycobacterium tuberculosis/genetics , Quality ControlABSTRACT
Phosphatidylinositides are one family of the most versatile signaling molecules in cells, yet how they interact with different proteins to regulate biological processes is not well understood. Towards a general strategy to identify phosphatidylinositide-protein interactions, a fluorous diazirine group has been incorporated into phosphatidylinositol 4,5-bisphosphate (PIP(2)). The modified PIP(2) was effectively cleaved by phospholipase C, one signaling protein that utilizes PIP(2) as its endogenous substrate. Upon light illumination, the PIP(2) probe effectively crosslinks with small GTPase ADP-ribosylation 1 to form a complex, suggesting that the probe might be suitable to identify PIP(2)-interacting proteins on the proteome level.
Subject(s)
ADP-Ribosylation Factor 1/chemistry , Fluorescent Dyes/chemistry , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositols/chemistry , ADP-Ribosylation Factor 1/analysis , Diazomethane , Molecular StructureABSTRACT
Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP(2) derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.
Subject(s)
Fluorescent Dyes/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Chromatography, Thin Layer/methods , Electrophoresis, Capillary/methods , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/chemistry , Kinetics , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphoinositide-3 Kinase Inhibitors , Sensitivity and SpecificityABSTRACT
To promote the coordinated development between renewable energy and the distribution network, a capacity allocation model of battery energy storage systems (BESS) is proposed to achieve the coordinated optimization for active and reactive power flow, which can reduce the voltage deviation and improve the absorptive capacity for renewable energy. In addition, BESS with four-quadrant operation characteristics, on-load tap changer, and capacitor banks are treated as flexible devices to improve the adaptability for renewable energy fluctuations. In view of the uncertainties of renewable energy caused by the inaccuracy of historical sample data, a set of extreme scenarios with the characteristics of temporal and spatial correlation are considered to obtain a robust BESS configuration decision. The big-M approach and the second-order conic relaxation technique are utilized to convert the BESS capacity allocation model into a mixed-integer linear programming problem. Finally, the IEEE 33-node distribution system is taken as an example to verify the effectiveness of the proposed method.
Subject(s)
Electric Power Supplies , Renewable EnergyABSTRACT
Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.
Subject(s)
Enzyme Assays/methods , Phosphatidylinositol 4,5-Diphosphate/analogs & derivatives , Type C Phospholipases/analysis , Fluorescein-5-isothiocyanate/chemistry , Hydrolysis , Isoenzymes/analysis , Lipid Metabolism/physiology , Lipids/chemistry , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phospholipase C gamma/analysis , Phospholipase C gamma/metabolism , Protein Binding/physiology , Type C Phospholipases/chemistry , Type C Phospholipases/metabolismABSTRACT
This study aims to investigate the microstructures, strength, and impact toughness of low-temperature bainite obtained by isothermal transformation at temperature below Ms (Martensite Starting temperature) for different times and tempering process in 0.53 C wt% bainitic steel. By using the optical microscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), electron back scatter diffraction (EBSD), and mechanical property test, it was found that the microstructures after heat treatment consist of small amounts of martensite, fine bainite, and film retained austenite. After tempered at 250 °C for 2 h, the volume fraction of retained austenite (10.9%) in the sample treated by isothermal transformation at 220 °C for three hours is almost the same as that of the sample without tempering. In addition, the retained austenite fraction decreases with the increase of holding times and is reduced to 6.8% after holding for 15 h. The ultimate tensile strength (1827 MPa), yield strength (1496 MPa), total elongations (16.1%), and impact toughness (up to 58 J/cm2) were obtained by isothermal transformation at 220 °C for three hours and tempered at 250 °C. Whereas, the impact toughness of sample without tempering is 28 J/cm2. After holding for 15 h, the impact toughness raises to 56 J/cm2, while the ductility and strength decreases. These results indicate that the tempering process is helpful to improve the impact toughness of low-temperature bainite.
ABSTRACT
OBJECTIVES: Most smokers begin using tobacco in their teens and recent reports indicate that smoking prevalence is climbing among youth in Taiwan. The purpose of this paper was to determine the associated factors of susceptibility of youth smoking by different types of schools. METHODS: A total of 4689 junior high students and 3918 senior high students participated in a school-based survey to determine the associated factors of susceptibility of youth smoking through anonymous self-administered questionnaire in 2004-2005. RESULTS: Susceptibility to initiate smoking ranged from 11.3% for junior high to 12.7% for general senior high and 12.4% for vocational senior students. For all/male smoking-susceptible students, more junior high students had one or more parents or best friends who smoked than did general or vocational senior high students. For all/female smoking-susceptible students, significantly more junior high students experienced secondhand smoke in public places than did non-susceptible students. CONCLUSIONS: Developing tailored, comprehensive smoking-prevention programs for junior high students should involve establishing tobacco-free households and communities.