This study investigated the effects of bromate (BrO3-), chlorite (NaClO2), and bromochloroacetic acid (BCAA) alone and in combination on renal cells. Exposure to BrO3- alone induced DNA damage, cell cycle arrest, cell death, and stress response protein expression. The addition of NaClO2 or BCAA increased BrO3--induced DNA damage and apoptosis, decreased necrosis, and altered stress protein expression. Exposure to all three chemicals together caused the greatest increases in DNA damage, apoptosis, cell cycle effects, and cell death along with decreases in ATP levels. The results suggest that water disinfection byproducts can interact synergistically to increase renal toxicity.
Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
The bc1 complex provides reduced cytochrome c to either the aa3 or cbb3 oxidases depending on oxygen conditions in R. sphaeroides. Strain BC-17 cannot photosynthesize due to lack of bc1 complex reducing cbb3. Some H217 mutations in the QI site can photosynthesize with DMSO but revert or are lethal without it. Future work should introduce H217 mutations into a DorR-/PpsR- background to uncouple effects on bc1 from changes to photosystem expression levels.
The document discusses the preparation and characterization of berberine-loaded bovine serum albumin (BSA) nanoparticles for efficient delivery of the anti-cancer drug berberine. Key points:
- Berberine-loaded BSA nanoparticles were prepared using a desolvation method and characterized using techniques like DLS, FESEM, FTIR, DSC and TGA.
- The nanoparticles were spherical in shape and monodisperse with a size of around 166 nm. FTIR confirmed encapsulation of berberine in the BSA nanoparticles.
- In vitro studies showed the nanoparticles had an entrapment efficiency of 85.65% and loading capacity of 7.78%. They provided sustained
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
This document contains 50 multiple choice questions related to biology, chemistry, and genetics. The questions cover a range of topics including enzyme kinetics, genetics, cellular processes, and metabolic pathways. For each question, four answer options are provided and only one answer is correct. All 50 questions must be answered.
This thesis investigates the presence of a carbonic anhydrase enzyme in the carboxysomes of the cyanobacterium Anabaena sp. PCC7120. Bioinformatic analysis identified a potential γ-class carbonic anhydrase, CcmM, based on sequence and structural similarity to known γ-CAs. Experiments showed CA activity in carboxysome extracts from Anabaena and other cyanobacteria. Recombinant CcmM was inactive but a truncated version, CcmM209, showed CA activity when bound to an affinity matrix or treated with an oxidizing agent, suggesting CcmM is a functional γ-CA regulated by its C-terminal domain. This work provides evidence
Assay Virtual Screening Compounds for the Inhibitory Potencies against BACE 1Ben Leong
This document describes a study conducted by three students to assay virtual screening compounds for their inhibitory potencies against BACE1, the enzyme involved in the production of amyloid beta peptides in Alzheimer's disease. Ten organic chemical compounds were tested for their half-maximal inhibitory concentration (IC50) values against BACE1 using a fluorescence resonance energy transfer assay. Compounds were then tested for toxicity on SH-SY5Y human neuroblastoma cells using the MTT assay. Six compounds showed potent micro-molar inhibition of BACE1, and some exhibited low cytotoxicity. Compound 7 had an IC50 of 4.49 μM against BACE1 and was not toxic to SH-SY5Y cells even at 10 μM
1) The document summarizes research into genetic interactors of the BRCA2 tumor suppressor gene, which is linked to hereditary breast cancer. A retroviral screening identified BRE as a genetic interactor that rescues lethality in BRCA2-deficient cells.
2) Further experiments showed that BRE overexpression in BRCA2-deficient cells leads to increased levels of the Cdc25A cell cycle regulator after DNA damage, preventing cell cycle arrest.
3) BRE was found to interact with the transcription factor ATF3 and induce transcription of Cdc25A. Reporter assays aim to identify the role of ATF3 binding sites on the Cdc25A promoter in regulating its transcription
Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
The bc1 complex provides reduced cytochrome c to either the aa3 or cbb3 oxidases depending on oxygen conditions in R. sphaeroides. Strain BC-17 cannot photosynthesize due to lack of bc1 complex reducing cbb3. Some H217 mutations in the QI site can photosynthesize with DMSO but revert or are lethal without it. Future work should introduce H217 mutations into a DorR-/PpsR- background to uncouple effects on bc1 from changes to photosystem expression levels.
The document discusses the preparation and characterization of berberine-loaded bovine serum albumin (BSA) nanoparticles for efficient delivery of the anti-cancer drug berberine. Key points:
- Berberine-loaded BSA nanoparticles were prepared using a desolvation method and characterized using techniques like DLS, FESEM, FTIR, DSC and TGA.
- The nanoparticles were spherical in shape and monodisperse with a size of around 166 nm. FTIR confirmed encapsulation of berberine in the BSA nanoparticles.
- In vitro studies showed the nanoparticles had an entrapment efficiency of 85.65% and loading capacity of 7.78%. They provided sustained
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
This document contains 50 multiple choice questions related to biology, chemistry, and genetics. The questions cover a range of topics including enzyme kinetics, genetics, cellular processes, and metabolic pathways. For each question, four answer options are provided and only one answer is correct. All 50 questions must be answered.
This thesis investigates the presence of a carbonic anhydrase enzyme in the carboxysomes of the cyanobacterium Anabaena sp. PCC7120. Bioinformatic analysis identified a potential γ-class carbonic anhydrase, CcmM, based on sequence and structural similarity to known γ-CAs. Experiments showed CA activity in carboxysome extracts from Anabaena and other cyanobacteria. Recombinant CcmM was inactive but a truncated version, CcmM209, showed CA activity when bound to an affinity matrix or treated with an oxidizing agent, suggesting CcmM is a functional γ-CA regulated by its C-terminal domain. This work provides evidence
Assay Virtual Screening Compounds for the Inhibitory Potencies against BACE 1Ben Leong
This document describes a study conducted by three students to assay virtual screening compounds for their inhibitory potencies against BACE1, the enzyme involved in the production of amyloid beta peptides in Alzheimer's disease. Ten organic chemical compounds were tested for their half-maximal inhibitory concentration (IC50) values against BACE1 using a fluorescence resonance energy transfer assay. Compounds were then tested for toxicity on SH-SY5Y human neuroblastoma cells using the MTT assay. Six compounds showed potent micro-molar inhibition of BACE1, and some exhibited low cytotoxicity. Compound 7 had an IC50 of 4.49 μM against BACE1 and was not toxic to SH-SY5Y cells even at 10 μM
1) The document summarizes research into genetic interactors of the BRCA2 tumor suppressor gene, which is linked to hereditary breast cancer. A retroviral screening identified BRE as a genetic interactor that rescues lethality in BRCA2-deficient cells.
2) Further experiments showed that BRE overexpression in BRCA2-deficient cells leads to increased levels of the Cdc25A cell cycle regulator after DNA damage, preventing cell cycle arrest.
3) BRE was found to interact with the transcription factor ATF3 and induce transcription of Cdc25A. Reporter assays aim to identify the role of ATF3 binding sites on the Cdc25A promoter in regulating its transcription
This study aimed to synthesize a library of 90 bromodomain inhibitors using a dihydropteridinone scaffold and cap-scanning technology. The inhibitors were tested for activity against BRD4 and BRDT using ALPHA screening. Several derivatives were identified with improved activity over the parent compound. Future work includes synthesizing inhibitors with different scaffolds and optimizing existing hits for non-BET bromodomain activity.
Protein corona associated with nanoparticlesANJUNITHIKURUP
The document discusses protein coronas that form around nanoparticles when introduced into biological fluids and how they impact physiological response. It summarizes that nanoparticles interact with proteins to form complexes with unique identities compared to the original nanoparticle. These complexes determine responses like uptake, circulation and toxicity. The document then examines several studies that show how nanoparticle properties like size and surface chemistry influence protein adsorption and subsequently impact biological response. It also reviews techniques for characterizing and experimentally investigating protein coronas and their effects.
This document summarizes research on developing polyrotaxane-based macromolecules as potential therapeutics for Niemann-Pick type C disease. Key points:
1) Polyrotaxanes consisting of 2-hydroxypropyl-β-cyclodextrin threaded onto Pluronic copolymers were synthesized to improve drug persistence and reduce required dosages.
2) Incorporating sulfobutyl ether-β-cyclodextrin improved water solubility.
3) The polyrotaxanes showed potential as MRI contrast agents by providing over 100-fold vascular enhancement compared to monomeric controls, circulating over 30 minutes with high relaxivity.
Overcoming challenges of host cell DNA removal in vaccine manufacturingDr. Priyabrata Pattnaik
Regulatory agencies require residual host cell DNA in vaccines to be extremely low, typically below 10 pg/dose. Various methods are used to remove DNA during vaccine manufacturing, including nuclease treatment, adsorptive depth filtration, chromatography, and tangential flow filtration. Nuclease treatment with Benzonase is widely used to digest DNA but the nuclease then needs to be removed using techniques like anion exchange chromatography, gel filtration, or ultrafiltration with diafiltration to achieve over 99% clearance.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
This document is a thesis submitted by Safwat Mohammad Abdul Azeez Saleh to An-Najah National University in partial fulfillment of the requirements for a Master of Science degree in Chemistry. The thesis investigates the HPLC determination and degradation kinetics of four textile dyes (Direct Red 81, Direct Blue 15, Direct Black 22, and Direct Orange 34). The study examines the effects of various parameters on dye degradation rates using iron and aluminum, including metal amount, dye concentration, temperature, pH, and agitation speed. The results provide insight into dye degradation mechanisms and allow for the quantitative analysis of dyes in real water samples using HPLC.
2014. Pietkiewicz, Wahyudi. Synthesis of macrocycles that inhibit protein syn...Adrian Pietkiewicz
This document describes the synthesis of seven new analogues of the macrocyclic peptide sanguinamide B (SanB) and testing of their ability to inhibit protein synthesis in cancer cells. The analogues were designed by altering the amino acids at positions I and III of the SanB backbone, inverting stereochemistry at position III, and changing the protecting group on lysine. All analogues were tested for cytotoxicity against colon cancer cell lines and ability to inhibit protein synthesis. The lead compound with an IC50 of 15.9 μM against colon cancer cells contained an N6-carboxybenzyl-lysine at position I. This establishes the importance of this moiety for biological activity.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
This document summarizes a Ph.D. thesis defense presentation by Hina Khalid from the Department of Bioinformatics and Biotechnology at GC University Faisalabad. The thesis involved an in silico and experimental investigation of synthetic compounds against Hepatitis C Virus. The presentation covered the need for the project, research objectives, methodology, and outcomes. The objectives were to develop assays to identify synthetic compounds with antiviral potential against HCV and minimal side effects, validate compounds' ability to inhibit the HCV polymerase NS5B, and characterize hit compounds' ability to restrict HCV replication in vitro. The methodology involved molecular virology experiments, compound preparation and screening, and computational modeling including molecular docking and dynamics simulations. Two potential hit
The document describes the development of the protein inhibitor AP24534 (Ponatinib) for treatment of chronic myeloid leukemia (CML). AP24534 was shown to inhibit the BCR-ABL protein, including the drug-resistant T315I mutation. Crystallography analysis revealed that AP24534 binds to BCR-ABL in the inactive conformation and maintains similar protein contacts as Imatinib. A phase I clinical trial found that AP24534 was selectively toxic to CML cells and active against T315I.
The document discusses using MALDI-TOF mass spectrometry to identify bacteria through protein biomarkers. MALDI-TOF allows analysis of intact bacterial cells by generating species-specific fingerprints from cell envelope components. These fingerprints can be entered into a database to rapidly identify unknown bacterial isolates.
1) The document evaluates the use of 5 E.coli biosensors containing the lux operon to test the preservative efficacy of sorbic acid according to pharmacopeia standards. 2) Results showed the bioluminescent readings of the biosensors correlated highly with colony forming unit counts (R^2 values >0.7) and detected ≥3 log reduction at similar times. 3) The lpp biosensor showed the greatest light reduction in response to sorbic acid exposure, while the spc and ldc biosensors best matched the ≥3 log reduction criteria, suggesting the method could replace traditional plate counts.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
The document describes a study analyzing the karyotype structure of Brachypodium stacei (2n=20) using comparative chromosome painting with Brachypodium distachyon as a reference. BAC clones from B. distachyon were selected and labeled as probes for fluorescence in situ hybridization to the chromosomes of B. stacei. Initial results showed the probes localized to specific loci on B. stacei chromosomes but also showed some nonspecific binding. Further work is needed to increase probe specificity to allow accurate analysis of the B. stacei karyotype and chromosome rearrangements compared to B. distachyon.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
This document provides an overview of molecular biology presented by Aaser Abdelazim. It discusses topics such as nucleic acid structures and functions, DNA replication, transcription, translation, mutations and repair. It also describes gene expression regulation and applications of molecular biology. Additionally, it covers molecular organization of the cell, what is molecular biology, gene concepts, DNA and RNA structures, DNA replication, transcription and translation processes. The document is intended as teaching material for molecular biology.
Effect of mixed species biofilm on corrosion of cast ironferibman
This document outlines an experimental study on the effect of mixed bacterial biofilms on corrosion of cast iron. The study aims to evaluate corrosion under mixed biofilms of denitrifying Pseudomonas aeruginosa and sulfate-reducing bacteria compared to individual biofilms. Materials and methods describe culturing the bacteria, preparing cast iron coupons, and electrochemical techniques like EIS and potentiodynamic polarization to characterize corrosion both with biofilms intact and after their removal. Preliminary results show the mixed biofilm reduces corrosion compared to sulfate-reducing bacteria alone, possibly due to interactions between denitrification and sulfate reduction pathways. Further experiments are planned to quantify biofilms and observe morphologies.
Rituxan mediates B-cell depletion through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Rituxan is not fully effective on its own and human serum enhances its potency. A model was developed showing that CDC, ADCC, and a serum augmentation of ADCC (SAM) act cooperatively rather than competitively. The synergy between these mechanisms was quantified, with human serum augmenting ADCC by 1.63-fold on average. Sensitivity analysis revealed that lysis is most sensitive to changes in CD20 expression levels and ADCC efficiency for B-cells expressing intermediate CD20 levels typical of Waldenstrom Macroglobulinemia.
This document describes experiments to produce and characterize the D4 protein binding domain of Clostridium botulinum toxin. The D4 protein was expressed in E. coli and purified using glutathione resin. It was then incubated with N2A cells that had been transfected with baculovirus to label early endosomes. Confocal microscopy showed that Alexa Fluor-labeled D4 protein bound to the endosomes of the N2A cells in a concentration-dependent manner.
The document discusses several key theories in landscape ecology, including percolation theory, source-sink models, and organism-space interactions. Percolation theory examines how the connectivity of occupied spaces in a landscape affects the movement of species. Source-sink models define certain areas as "sources" that positively contribute to ecological processes or "sinks" that are unhelpful. These concepts can be applied to issues like wildfire spread, disease transmission, and resource usage. Organism-space interaction theories explore how population dynamics depend on landscape characteristics such as habitat size, proximity, and fragmentation.
This document discusses disinfection byproducts (DBPs) formed when disinfectants like chlorine react with organic matter in water. It covers the classification and factors influencing DBP formation, and the health impacts of several DBPs including increased risks of various cancers, cardiovascular disease, central nervous system effects, and adverse pregnancy outcomes. Specific DBPs are linked to thyroid cancer, lung cancer, lymphoma, liver cancer, bladder cancer, skin cancer, and impacts on the central nervous system and pregnancy. The document provides an introduction to DBPs and their role in water disinfection.
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This study aimed to synthesize a library of 90 bromodomain inhibitors using a dihydropteridinone scaffold and cap-scanning technology. The inhibitors were tested for activity against BRD4 and BRDT using ALPHA screening. Several derivatives were identified with improved activity over the parent compound. Future work includes synthesizing inhibitors with different scaffolds and optimizing existing hits for non-BET bromodomain activity.
Protein corona associated with nanoparticlesANJUNITHIKURUP
The document discusses protein coronas that form around nanoparticles when introduced into biological fluids and how they impact physiological response. It summarizes that nanoparticles interact with proteins to form complexes with unique identities compared to the original nanoparticle. These complexes determine responses like uptake, circulation and toxicity. The document then examines several studies that show how nanoparticle properties like size and surface chemistry influence protein adsorption and subsequently impact biological response. It also reviews techniques for characterizing and experimentally investigating protein coronas and their effects.
This document summarizes research on developing polyrotaxane-based macromolecules as potential therapeutics for Niemann-Pick type C disease. Key points:
1) Polyrotaxanes consisting of 2-hydroxypropyl-β-cyclodextrin threaded onto Pluronic copolymers were synthesized to improve drug persistence and reduce required dosages.
2) Incorporating sulfobutyl ether-β-cyclodextrin improved water solubility.
3) The polyrotaxanes showed potential as MRI contrast agents by providing over 100-fold vascular enhancement compared to monomeric controls, circulating over 30 minutes with high relaxivity.
Overcoming challenges of host cell DNA removal in vaccine manufacturingDr. Priyabrata Pattnaik
Regulatory agencies require residual host cell DNA in vaccines to be extremely low, typically below 10 pg/dose. Various methods are used to remove DNA during vaccine manufacturing, including nuclease treatment, adsorptive depth filtration, chromatography, and tangential flow filtration. Nuclease treatment with Benzonase is widely used to digest DNA but the nuclease then needs to be removed using techniques like anion exchange chromatography, gel filtration, or ultrafiltration with diafiltration to achieve over 99% clearance.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
This document is a thesis submitted by Safwat Mohammad Abdul Azeez Saleh to An-Najah National University in partial fulfillment of the requirements for a Master of Science degree in Chemistry. The thesis investigates the HPLC determination and degradation kinetics of four textile dyes (Direct Red 81, Direct Blue 15, Direct Black 22, and Direct Orange 34). The study examines the effects of various parameters on dye degradation rates using iron and aluminum, including metal amount, dye concentration, temperature, pH, and agitation speed. The results provide insight into dye degradation mechanisms and allow for the quantitative analysis of dyes in real water samples using HPLC.
2014. Pietkiewicz, Wahyudi. Synthesis of macrocycles that inhibit protein syn...Adrian Pietkiewicz
This document describes the synthesis of seven new analogues of the macrocyclic peptide sanguinamide B (SanB) and testing of their ability to inhibit protein synthesis in cancer cells. The analogues were designed by altering the amino acids at positions I and III of the SanB backbone, inverting stereochemistry at position III, and changing the protecting group on lysine. All analogues were tested for cytotoxicity against colon cancer cell lines and ability to inhibit protein synthesis. The lead compound with an IC50 of 15.9 μM against colon cancer cells contained an N6-carboxybenzyl-lysine at position I. This establishes the importance of this moiety for biological activity.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
This document summarizes a Ph.D. thesis defense presentation by Hina Khalid from the Department of Bioinformatics and Biotechnology at GC University Faisalabad. The thesis involved an in silico and experimental investigation of synthetic compounds against Hepatitis C Virus. The presentation covered the need for the project, research objectives, methodology, and outcomes. The objectives were to develop assays to identify synthetic compounds with antiviral potential against HCV and minimal side effects, validate compounds' ability to inhibit the HCV polymerase NS5B, and characterize hit compounds' ability to restrict HCV replication in vitro. The methodology involved molecular virology experiments, compound preparation and screening, and computational modeling including molecular docking and dynamics simulations. Two potential hit
The document describes the development of the protein inhibitor AP24534 (Ponatinib) for treatment of chronic myeloid leukemia (CML). AP24534 was shown to inhibit the BCR-ABL protein, including the drug-resistant T315I mutation. Crystallography analysis revealed that AP24534 binds to BCR-ABL in the inactive conformation and maintains similar protein contacts as Imatinib. A phase I clinical trial found that AP24534 was selectively toxic to CML cells and active against T315I.
The document discusses using MALDI-TOF mass spectrometry to identify bacteria through protein biomarkers. MALDI-TOF allows analysis of intact bacterial cells by generating species-specific fingerprints from cell envelope components. These fingerprints can be entered into a database to rapidly identify unknown bacterial isolates.
1) The document evaluates the use of 5 E.coli biosensors containing the lux operon to test the preservative efficacy of sorbic acid according to pharmacopeia standards. 2) Results showed the bioluminescent readings of the biosensors correlated highly with colony forming unit counts (R^2 values >0.7) and detected ≥3 log reduction at similar times. 3) The lpp biosensor showed the greatest light reduction in response to sorbic acid exposure, while the spc and ldc biosensors best matched the ≥3 log reduction criteria, suggesting the method could replace traditional plate counts.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
The document describes a study analyzing the karyotype structure of Brachypodium stacei (2n=20) using comparative chromosome painting with Brachypodium distachyon as a reference. BAC clones from B. distachyon were selected and labeled as probes for fluorescence in situ hybridization to the chromosomes of B. stacei. Initial results showed the probes localized to specific loci on B. stacei chromosomes but also showed some nonspecific binding. Further work is needed to increase probe specificity to allow accurate analysis of the B. stacei karyotype and chromosome rearrangements compared to B. distachyon.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
This document provides an overview of molecular biology presented by Aaser Abdelazim. It discusses topics such as nucleic acid structures and functions, DNA replication, transcription, translation, mutations and repair. It also describes gene expression regulation and applications of molecular biology. Additionally, it covers molecular organization of the cell, what is molecular biology, gene concepts, DNA and RNA structures, DNA replication, transcription and translation processes. The document is intended as teaching material for molecular biology.
Effect of mixed species biofilm on corrosion of cast ironferibman
This document outlines an experimental study on the effect of mixed bacterial biofilms on corrosion of cast iron. The study aims to evaluate corrosion under mixed biofilms of denitrifying Pseudomonas aeruginosa and sulfate-reducing bacteria compared to individual biofilms. Materials and methods describe culturing the bacteria, preparing cast iron coupons, and electrochemical techniques like EIS and potentiodynamic polarization to characterize corrosion both with biofilms intact and after their removal. Preliminary results show the mixed biofilm reduces corrosion compared to sulfate-reducing bacteria alone, possibly due to interactions between denitrification and sulfate reduction pathways. Further experiments are planned to quantify biofilms and observe morphologies.
Rituxan mediates B-cell depletion through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Rituxan is not fully effective on its own and human serum enhances its potency. A model was developed showing that CDC, ADCC, and a serum augmentation of ADCC (SAM) act cooperatively rather than competitively. The synergy between these mechanisms was quantified, with human serum augmenting ADCC by 1.63-fold on average. Sensitivity analysis revealed that lysis is most sensitive to changes in CD20 expression levels and ADCC efficiency for B-cells expressing intermediate CD20 levels typical of Waldenstrom Macroglobulinemia.
This document describes experiments to produce and characterize the D4 protein binding domain of Clostridium botulinum toxin. The D4 protein was expressed in E. coli and purified using glutathione resin. It was then incubated with N2A cells that had been transfected with baculovirus to label early endosomes. Confocal microscopy showed that Alexa Fluor-labeled D4 protein bound to the endosomes of the N2A cells in a concentration-dependent manner.
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The document discusses several key theories in landscape ecology, including percolation theory, source-sink models, and organism-space interactions. Percolation theory examines how the connectivity of occupied spaces in a landscape affects the movement of species. Source-sink models define certain areas as "sources" that positively contribute to ecological processes or "sinks" that are unhelpful. These concepts can be applied to issues like wildfire spread, disease transmission, and resource usage. Organism-space interaction theories explore how population dynamics depend on landscape characteristics such as habitat size, proximity, and fragmentation.
This document discusses disinfection byproducts (DBPs) formed when disinfectants like chlorine react with organic matter in water. It covers the classification and factors influencing DBP formation, and the health impacts of several DBPs including increased risks of various cancers, cardiovascular disease, central nervous system effects, and adverse pregnancy outcomes. Specific DBPs are linked to thyroid cancer, lung cancer, lymphoma, liver cancer, bladder cancer, skin cancer, and impacts on the central nervous system and pregnancy. The document provides an introduction to DBPs and their role in water disinfection.
T2 Laboratories experienced a chemical explosion in 2007 due to a runaway exothermic reaction during the production of MCMT. The explosion was caused by a loss of sufficient cooling during the process, which led to an uncontrollable increase in pressure and temperature inside the reactor. The explosion damaged the facility equivalent to 1400 pounds of TNT. The study found that T2 Laboratories lacked proper process safety information and hazard analysis, did not understand the chemistry's potential for runaway reactions, and had an improperly designed emergency relief system. The incident highlights the importance of thoroughly understanding chemical reactions and implementing safety systems, such as cooling and pressure controls, when scaling up chemical production.
This document discusses India's legislative efforts to regulate ethical guidelines for biomedical research involving human subjects. It provides background on India's existing codes of ethics and guidelines. It then summarizes the process by which India developed its 2000 Ethical Guidelines for Biomedical Research, including establishing expert committees. Major issues addressed in the 2000 guidelines are outlined. The document notes some instances of unethical clinical trials in India and the need for laws to protect research participants. It proposes the Biomedical Research on Human Subjects Bill to establish a regulatory authority and oversight mechanisms like an ethics committee system. The functions and composition of the proposed authority are described.
Acid rain is caused by emissions of sulfur and nitrogen oxides from fossil fuel combustion. It damages sensitive ecosystems through acidification of soils and freshwater bodies. The key effects are leaching of nutrients from soils, release of aluminum which is toxic to plants and fish, and loss of biodiversity in freshwater systems. The Clean Air Act established a two-phase program in the 1990s that significantly reduced sulfur dioxide emissions from power plants in the eastern U.S., achieving over a 50% reduction in related acid rain. However, developing accurate source-receptor models to guide regulation remains an ongoing challenge.
This document discusses disinfection byproducts (DBPs) formed when disinfectants like chlorine react with organic matter in water. It covers the classification and factors influencing DBP formation, and the health impacts of several DBPs including increased risks of various cancers, cardiovascular disease, central nervous system effects, and adverse pregnancy outcomes. Specific DBPs are linked to thyroid cancer, lung cancer, lymphoma, liver cancer, bladder cancer, skin cancer, and impacts on the central nervous system and pregnancy. The document provides an introduction to DBPs and their role in water disinfection.
This document discusses various types and sources of water pollution. It identifies major pollutants like infectious agents, oxygen-demanding waste, and inorganic chemicals that come from sources such as sewage, industry, and agriculture. These pollutants can contaminate both surface water and groundwater, and have negative effects on ecosystems and human health. The document also examines solutions for preventing and treating water pollution through various regulatory, technological, and natural approaches.
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Epcon is One of the World's leading Manufacturing Companies. With over 4000 installations worldwide, EPCON has been pioneering new techniques since 1977 that have become industry standards now. Founded in 1977, Epcon has grown from a one-man operation to a global leader in developing and manufacturing innovative air pollution control technology and industrial heating equipment.
Optimizing Post Remediation Groundwater Performance with Enhanced Microbiolog...Joshua Orris
Results of geophysics and pneumatic injection pilot tests during 2003 – 2007 yielded significant positive results for injection delivery design and contaminant mass treatment, resulting in permanent shut-down of an existing groundwater Pump & Treat system.
Accessible source areas were subsequently removed (2011) by soil excavation and treated with the placement of Emulsified Vegetable Oil EVO and zero-valent iron ZVI to accelerate treatment of impacted groundwater in overburden and weathered fractured bedrock. Post pilot test and post remediation groundwater monitoring has included analyses of CVOCs, organic fatty acids, dissolved gases and QuantArray® -Chlor to quantify key microorganisms (e.g., Dehalococcoides, Dehalobacter, etc.) and functional genes (e.g., vinyl chloride reductase, methane monooxygenase, etc.) to assess potential for reductive dechlorination and aerobic cometabolism of CVOCs.
In 2022, the first commercial application of MetaArray™ was performed at the site. MetaArray™ utilizes statistical analysis, such as principal component analysis and multivariate analysis to provide evidence that reductive dechlorination is active or even that it is slowing. This creates actionable data allowing users to save money by making important site management decisions earlier.
The results of the MetaArray™ analysis’ support vector machine (SVM) identified groundwater monitoring wells with a 80% confidence that were characterized as either Limited for Reductive Decholorination or had a High Reductive Reduction Dechlorination potential. The results of MetaArray™ will be used to further optimize the site’s post remediation monitoring program for monitored natural attenuation.
Presented by The Global Peatlands Assessment: Mapping, Policy, and Action at GLF Peatlands 2024 - The Global Peatlands Assessment: Mapping, Policy, and Action
Microbial characterisation and identification, and potability of River Kuywa ...Open Access Research Paper
Water contamination is one of the major causes of water borne diseases worldwide. In Kenya, approximately 43% of people lack access to potable water due to human contamination. River Kuywa water is currently experiencing contamination due to human activities. Its water is widely used for domestic, agricultural, industrial and recreational purposes. This study aimed at characterizing bacteria and fungi in river Kuywa water. Water samples were randomly collected from four sites of the river: site A (Matisi), site B (Ngwelo), site C (Nzoia water pump) and site D (Chalicha), during the dry season (January-March 2018) and wet season (April-July 2018) and were transported to Maseno University Microbiology and plant pathology laboratory for analysis. The characterization and identification of bacteria and fungi were carried out using standard microbiological techniques. Nine bacterial genera and three fungi were identified from Kuywa river water. Clostridium spp., Staphylococcus spp., Enterobacter spp., Streptococcus spp., E. coli, Klebsiella spp., Shigella spp., Proteus spp. and Salmonella spp. Fungi were Fusarium oxysporum, Aspergillus flavus complex and Penicillium species. Wet season recorded highest bacterial and fungal counts (6.61-7.66 and 3.83-6.75cfu/ml) respectively. The results indicated that the river Kuywa water is polluted and therefore unsafe for human consumption before treatment. It is therefore recommended that the communities to ensure that they boil water especially for drinking.
Evolving Lifecycles with High Resolution Site Characterization (HRSC) and 3-D...Joshua Orris
The incorporation of a 3DCSM and completion of HRSC provided a tool for enhanced, data-driven, decisions to support a change in remediation closure strategies. Currently, an approved pilot study has been obtained to shut-down the remediation systems (ISCO, P&T) and conduct a hydraulic study under non-pumping conditions. A separate micro-biological bench scale treatability study was competed that yielded positive results for an emerging innovative technology. As a result, a field pilot study has commenced with results expected in nine-twelve months. With the results of the hydraulic study, field pilot studies and an updated risk assessment leading site monitoring optimization cost lifecycle savings upwards of $15MM towards an alternatively evolved best available technology remediation closure strategy.
Improving the viability of probiotics by encapsulation methods for developmen...Open Access Research Paper
The popularity of functional foods among scientists and common people has been increasing day by day. Awareness and modernization make the consumer think better regarding food and nutrition. Now a day’s individual knows very well about the relation between food consumption and disease prevalence. Humans have a diversity of microbes in the gut that together form the gut microflora. Probiotics are the health-promoting live microbial cells improve host health through gut and brain connection and fighting against harmful bacteria. Bifidobacterium and Lactobacillus are the two bacterial genera which are considered to be probiotic. These good bacteria are facing challenges of viability. There are so many factors such as sensitivity to heat, pH, acidity, osmotic effect, mechanical shear, chemical components, freezing and storage time as well which affects the viability of probiotics in the dairy food matrix as well as in the gut. Multiple efforts have been done in the past and ongoing in present for these beneficial microbial population stability until their destination in the gut. One of a useful technique known as microencapsulation makes the probiotic effective in the diversified conditions and maintain these microbe’s community to the optimum level for achieving targeted benefits. Dairy products are found to be an ideal vehicle for probiotic incorporation. It has been seen that the encapsulated microbial cells show higher viability than the free cells in different processing and storage conditions as well as against bile salts in the gut. They make the food functional when incorporated, without affecting the product sensory characteristics.
ENVIRONMENT~ Renewable Energy Sources and their future prospects.tiwarimanvi3129
This presentation is for us to know that how our Environment need Attention for protection of our natural resources which are depleted day by day that's why we need to take time and shift our attention to renewable energy sources instead of non-renewable sources which are better and Eco-friendly for our environment. these renewable energy sources are so helpful for our planet and for every living organism which depends on environment.
Kinetic studies on malachite green dye adsorption from aqueous solutions by A...Open Access Research Paper
Water polluted by dyestuffs compounds is a global threat to health and the environment; accordingly, we prepared a green novel sorbent chemical and Physical system from an algae, chitosan and chitosan nanoparticle and impregnated with algae with chitosan nanocomposite for the sorption of Malachite green dye from water. The algae with chitosan nanocomposite by a simple method and used as a recyclable and effective adsorbent for the removal of malachite green dye from aqueous solutions. Algae, chitosan, chitosan nanoparticle and algae with chitosan nanocomposite were characterized using different physicochemical methods. The functional groups and chemical compounds found in algae, chitosan, chitosan algae, chitosan nanoparticle, and chitosan nanoparticle with algae were identified using FTIR, SEM, and TGADTA/DTG techniques. The optimal adsorption conditions, different dosages, pH and Temperature the amount of algae with chitosan nanocomposite were determined. At optimized conditions and the batch equilibrium studies more than 99% of the dye was removed. The adsorption process data matched well kinetics showed that the reaction order for dye varied with pseudo-first order and pseudo-second order. Furthermore, the maximum adsorption capacity of the algae with chitosan nanocomposite toward malachite green dye reached as high as 15.5mg/g, respectively. Finally, multiple times reusing of algae with chitosan nanocomposite and removing dye from a real wastewater has made it a promising and attractive option for further practical applications.
Climate Change All over the World .pptxsairaanwer024
Climate change refers to significant and lasting changes in the average weather patterns over periods ranging from decades to millions of years. It encompasses both global warming driven by human emissions of greenhouse gases and the resulting large-scale shifts in weather patterns. While climate change is a natural phenomenon, human activities, particularly since the Industrial Revolution, have accelerated its pace and intensity
3. Research back ground
Bromate (BrO3-) is a water disinfection by-product (DBP) and is possibly carcinogenic to humans.
One of the major target organs of BrO3- is the kidney.
Chronic exposure to high doses of BrO3- induces renal adenoma and carcinoma and non-cancerous urothelial hyperplasia. BrO3-
induced renal toxicity has been associated with DNA damage, primarily8-hydroxyl de oxy- guanosine (8-OHdG) formation.
Chlorite is a regulated water disinfection by-product found in g/l concentrations in some disinfected waters.
BCAA is currently an unregulated halo acid, which constitutes one of the most prevalent by-products in drinking water.
BCAA increased incidences of adenomas of the large intestine and in rats, and induced hepatocellular neoplasms and hepatoblastoma.
Non-neoplastic liver lesions were observed in BCAA exposure.
A gene array study suggested that carcinogenic pathways involved in BCAA induced mesothelioma include insulin-like growth factor 1
(IGF-1), p38 MAPK, Wnt/beta-catenin and integrin signaling pathways.
4. Research objectives
To investigate the effect of NaClO2 and BCAA on BrO3--induced DNA damage
and renal cell death.
To increase the understanding of the mode of these chemicals and increase the
knowledge on the mixture on renal toxicity.
5. Reagents and
antidotes
Cell culture
Assessment of
morphology
Assessment of
cell viability
Nuclear
morphology
Measurement of cell
death
Measurement of
cell cycles
Immunoblot
analysis
Immunocyto
chemistry
ROS
measurement
Measurement of
ATP levels
Protein Determination
Statistical
analysis
Experimental design
6. Results
Effect of NaClO2 and BCAA on BrO3--induced alterations in MTT
staining and cell morphology.
This study determined the time- and concentration-dependence
of BrO3- cytotoxicity, and showed that concentrations of BrO3- from
100 to 400 ppm induce necrosis in NRK cells after 48 h of
exposure. BrO3- (200 ppm) moderately decreased MTT staining in
NRK cells after 48 h.
Treatment of NRK cells with NaClO2 alone decreased MTT staining
only at concentrations over 20 ppm. In contrast, treatment of NRK
cells with 10 or 20 ppm NaClO2 simultaneously with BrO3
decreased MTT staining compared to cells exposed to BrO3 alone.
Exposure of NRK cells to only BCAA decreased MTT staining at
concentrations of 50 ppm and above. Treatment of cells with 50 ppm
BCAA simultaneously with BrO3- decreased MTT staining
compared to cells exposed to BrO3- alone.
Exposure of cells to 10 ppm NaClO2 and 50 ppm BCAA
simultaneously with BrO3 significantly decreased MTT staining
compared to cells exposed BrO3- alone.
In contrast, exposure of cells to a mixture
of just NaClO2 and BCAA together resulted in slight decreases in
MTT staining. The effects of NaClO2 and BCAA on BrO3—induced
decreases in MTT staining were confirmed by cell morphology
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG formation. NRK cells were treated with 200 ppm KBrO3 alone, or in
combination with NaClO2, BCAA or both for 24 h prior to analysis of nuclear morphology and 8-OHdG staining using fluorescence
microscopy. Representative staining for DAPI (shown in blue) and 8-OHdG (shown in green) is depicted in (A) and the percentage
of nuclei staining positive for 8-OHdG after exposure to 100 or 200 ppm BrO3- are shown in (B and C). White arrows in (A) ndicate
nuclei staining positive for 8-OHdG. Data are represented as the mean ± SEM of at least 3 separate experiments. Means with
different subscripts are significantly (P < 0.05) different from each other.
7. Results
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG
staining.
Treatment of NRK cells with 100 or 200 ppm BrO3-
alone induced moderate increases in 8 OHdG staining
as assessed by fluorescence microscopy.
Exposure of cells to all three DBPs increased 8-OHdG
staining to levels comparable to cells exposed to both
BrO3- and NaClO2.
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG formation. NRK cells were treated with 200 ppm KBrO3 alone, or in
combination with NaClO2, BCAA or both for 24 h prior to analysis of nuclear morphology and 8-OHdG staining using fluorescence
microscopy. Representative staining for DAPI (shown in blue) and 8-OHdG (shown in green) is depicted in (A) and the percentage of
nuclei staining positive for 8-OHdG after exposure to 100 or 200 ppm BrO3- are shown in (B and C). White arrows in (A)
indicate nuclei staining positive for 8-OHdG. Data are represented as the mean ± SEM of at least 3 separate experiments.
8. Results
Effect of NaClO2 and BCAA on BrO3--induced
alterations in cell cycle.
Exposure to NaClO2 and BCAA alters the cell cycle
arrest induced by BrO3-. Treatment of cells with NaClO2
(10–40 ppm) alone induced a moderate G2/M arrest.
BCAA exposure only slightly altered NRK cell
cycle at concentrations as high as 50 ppm.
Exposure of cells to NaClO2 and BrO3- significantly
decreased the percentage of cells in the G2/M phase of
the cell cycle, compared to cells exposed to
BrO3- alone.
BCAA had no effect on BrO3 induced changes in cell
cycle .
Exposure of cells to a mixture of all three chemicals
yielded results similar to that seen
in cells exposed to BrO3- and NaClO2.
Effect of NaClO2 and BCAA on BrO3--induced cell cycle arrest. NRK cells were treated with 10, 20 and 40 ppm
NaClO2 alone (A) or 20 and 50 ppm BCAA alone (B)for 24 h prior to analysis of cell cycle using PI staining and flow
cytometry. Cells were exposed to 200 ppm KBrO3 alone, or in combination with 20 ppm NaClO2 (C), 20 ppmBCAA
(D), or both (E) for 24 h, prior to analysis of cell cycle. Data are represented as the mean ± SEM of at least 3
separate experiments
9. Effect of NaClO2 and BCAA on BrO3--induced cell death.
we exposed NRK cells to these chemicals alone or in mixtures, and assessed alterations in annexin V
(apoptotic cell marker) and PI (necrotic cell marker) staining using flow cytometry.
Exposure of cells to BrO3- alone increased the percentage of cells staining positive for PI alone, without
increasing the number of cells staining positive for annexin V alone, or for both annexin V and PI (late
apoptosis).
Interestingly exposure to both BrO3- and NaClO2 decreased the percentage of cells staining positive for PI
alone, and increased the percentage of cell staining positive for both annexin V and PI.
Interestingly exposure to both BrO3- and NaClO2 decreased the percentage of cells staining positive for PI
alone, and increased the percentage of annexin V positive cells.
Exposure of cells to BCAA creased the percentage of cells staining positive for both annexin V and PI
ercentage of annexin V positive cells. and BrO3- also decreased the percentage of cells staining positive for
PI alone and increased the number of annexin V positive cells; however, BCAA also in BCAA also
increased the percentage of cells.
Treatment of cells with all three chemicals significantly increased the percentage of cells staining positive
for annexin V alone, and those staining positive for both annexin V and PI. These increases were greater
than those observed in cellsexposed to BrO3- and NaClO2, or BrO3- and BCA
Results
10. Treatment of cells with all three chemicals
significantly increased the percentage of cells
staining positive for annexin V alone, and those
staining positive for both annexin V and PI. These
increases were greater than those observed in cells
exposed to BrO3- and NaClO2, or BrO3- and
BCAA.
we assess DAPI staining and annexin and PI
staining using fluorescence microscopy.
Exposure of cells to BrO3-, NaClO2 and BCAA
resulted in an increase in cells staining positive
for annexin V, with the characteristic halo-like
pattern of apoptosis
Results
Effect of NaClO2 and BCAA on BrO3--induced cell death and nuclear morphology. NRK cells were exposed to 200 ppm KBrO3
alone or in combination with 10 ppm NaClO2 (A), 20 ppm BCAA (B) or both (C) for 48 h prior to analysis of annexin V and PI
staining as determined using flow cytometry. In addition, nuclear morphology (D, upper panel) and annexin and PI staining (D,
lower panel) were assessed using fluorescence microscopy. Cisplatin was used in (D) as a positive control. Data in (A–C) are
represented as the mean ± SEM of at least 3 separate experiments. The arrows represent apoptotic nuclear morphology (D, upper
panel) and the characteristic halo-like staining of annexin V-FITC of the plasma membrane (D, lower panel). Means with different
subscripts are significantly (P < 0.05) different from each other. Data in (D) are representative of at least 3 individual experiments
11. ClO2 and BCAA on BrO3--induced ROS
formation.
Exposure of cells to BrO3 alone increased CM-
H2DCFDA staining compared to control cells.
Exposure of cells to NaClO2 and BCAA alone
also increased CM-H2DCFDA staining.
Exposure of cells to BrO3 and NaClO2, or BrO3-
and BCAA, or a mixture of all three, did not
significantly increase CM-H2DCFDA staining
compared to cells exposed to BrO3- alone.
It suggest that the ability of mixtures of DBPs to
increase cell death is not a result in an increase
in the overall levels of ROS.
Results
Effect of NaClO2 and BCAA on BrO3--induced ROS formation. NRK cells were preloaded with 10 M CM-H2DCFDA for 30 min and then
exposed to NaClO2 (0–50 ppm) (A), BCAA (0–150 ppm) (B) or 200 ppm KBrO3 alone, or in combination with 10 ppm NaClO2 alone, 20
ppm BCAA alone or both (C) for 30 min prior to measurement of fluorescence intensity. Data are represented as the mean ± SEM of at
least 3 separate experiments. Means with different subscripts are significantly (P < 0.05) different from each other.
12. Effect of NaClO2 and BCAA on BrO3--
induced expression of DNA damage and
stress response proteins
Exposure of cells to BrO3- alone increased the
expression of numerous stress response and DNA
damage response proteins, including p38 MAPK
and p21, as well as H2AX phosphorylation
and p53 phosphorylation.
Exposure of cells to all three chemicals increased
the expression of p-p38 compared to BrO3-
alone.
Interestingly, p-p53 expression was similar to
that seen in cells exposed to BrO3- alone, while
the expression of p21 and p-H2AX were
essentially unchanged.
Results
Effect of NaClO2 and BCAA on BrO3--induced alterations in cell signaling protein expression. NRK cells were exposed to 200 ppm
KBrO3 alone, or in combination with 10 ppm NaClO2, 20 ppm BCAA or both for 24 h prior to analysis of protein expression using
immunoblot analysis. p38 is shown as a loading control. All blots are representative of at least 3 separate experiment
13. Effect of NaClO2 and BCAA on BrO3 induced alterations
in ATP.
Treatment of cells with BrO3- alone induced
concentration dependent decreases in ATP levels.
BrO3- concentrations of 200 ppm decreased ATP
levels 20%, which is consistent with the level of
cell death induced at this concentration.
Treatment of cells with 10 ppm NaClO2 decreased
cellular ATP levels 20%, while treatment with 20
ppm BCAA had no significant affect, compared to
control cells.
In contrast, treatment of cells with mixtures
of all three chemicals decreased cellular ATP levels
60% compared to control cells.
Decrease in cellular ATP levels in the presence of
all three chemicals is consistent with decreases in
cell viability .
Results
Effect of NaClO2 and BCAA on BrO3--induced ATP depletion. NRK cells were exposed to 0–400 ppm KBrO3 (A) alone
for 48 h, or to 200 ppm KBrO3 in combination with 10 ppm NaClO2 or 200 ppm BCAA or both (B) for 48 h prior to
analysis of cellular ATP levels using luminescence assays. Data are represented as the mean ± SEM of at least 3
separate experiments
14. Proposed interactions between DBPs and renal cell
death. Exposure of renal cells to BrO3- alone
activates DNA damage-independent and DNA
damage dependent pathways. The DNA-independent
pathway involves a ROS-mediated MAPK pathway
in which p38 activates p53 and p21.
The DNA damage-dependent pathway results in 8-
OHdG formation, which also leads to p53 and p21
activation.
Exposure of cells to BrO3- in the presence of
NaClO2 increases 8-OHdG formation,
p53 activation and p21 expression, as well as H2AX
phosphorylation.
In contrast, exposure of cells to BrO3- in the
presence of BCAA increases p38 and p53 activation
and p21 expression, without increasing 8-OHdG
formation.
The interactions between these DBPs appear to
switch the mechanisms of BrO3--induced renal cell
death from necrosis to apoptosis.
Results
15. conclusion
DBPs synergistically increases cell death
Increases in cell death correlated to increased DNA damage, apoptosis,
alteration in the cell cycle as well as alterations in expression of cells
stress and DNA damage response proteins.