Concentrations Of Dithiothreitol Research Articles

Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence.

The malachite green aptamer (MGapt) is known for its utility in RNA measurement in vivo and in lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect RNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system's chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.

Supplementation with Different Concentrations of Dithiothreitol and Glutathione During Cryopreservation of Semen from Large White Boars

Supplementation with Different Concentrations of Dithiothreitol and Glutathione During Cryopreservation of Semen from Large White Boars

The effect of probe density coverage on the detection of oenological tannins in Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) experiments.

Polyphenols are a group of compounds found in grapes, musts, and wines. Their levels are crucial for grape ripening, proper must fermentation, and final wine characteristics. Standard chemical analysis is commonly used to detect these compounds, but it is costly, time-consuming, and requires specialized laboratories and operators. To address this, this study explores a functionalized acoustic sensor for detecting oenological polyphenols. The method involves utilizing a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to detect the target analyte by using a gelatin-based probe layer. The sensor is functionalized by optimizing the probe coverage density to maximize its performance. This is achieved by using 12-mercaptododecanoic acid (12-MCA) to immobilize the probe onto the gold sensor surface, and dithiothreitol (DTT) as a reducing and competitive binding agent. The concentration of 12-MCA and DTT in the solutions is varied to control the probe density. QCM-D measurements demonstrates that the probe density can be effectively adjusted using this approach, ranging from 0.2×1013 molecules cm-2 to 2×1013 molecules cm-2 . This study also investigates the interaction between the probe and tannins, confirming the sensor ability to detect them. Interestingly, the lower probe coverage achieves higher detection signals when normalized to probe immobilization signals. Moreover, significant changes in mechanical properties of the functionalization layer are observed after the interaction with samples. The combination of QCM-D with gelatin functionalization holds great promise for future applications in the wine industry. It offers real-time monitoring capabilities, requires minimal sample preparation, and provides high sensitivity for quality control purposes. This article is protected by copyright. All rights reserved.

Application of Modified DTT Methods to Reduce the Interference of Daratumumab on Serological Detection

To modified the classic dithiothreitol (DTT) method for treating red blood cells (RBCs) in Technical Manual of American Association of Blood Banks（AABB） and evaluate its application value in pre-transfusion examination of patients treated with daratumumab. The classic 0.2 mol/L DTT method was improved in terms of PBS, DTT concentration, donor RBCs concentration (suspended/packed) and sample processing time. The modified DTT methods and AABB classic DTT method were applied to the blood matching tests of 12 multiple myeloma patients treated with daratumumab. The effect of treating panel RBCs with modified DTT methods on the detection of other irregular antibodies was evaluated by using antiserum and antibody reagents with known antibody properties. Two modified DTT methods were established (method 1: changed the concentration of DTT to 0.01 mol/L; method 2: changed the concentration of DTT to 0.02 mol/L and replaced the packed RBCs with 3% RBCs suspension). The optimal treatment time was 35 min for the modified DTT methods. At this time, the pan-agglutination caused by daratumumab was eliminated, but the detection of antibodies such as anti-E, anti-JKa, anti-M were not affected, and the titer of anti-K antibodies was only slightly decreased. The modified DTT methods were effective, which can eliminate the interference of daratumumab while retaining the activity of the Kell blood group system, and can replace the current classic DTT method in AABB Technical Manual.

Smart transformation of bowl shape chitosan nanomotors to disc shape in simulated biological media and consequent controlled velocity

The design of nanomotors appropriate for targeted drug delivery to cancer cells have been extensively investigated in recent years. By Pickering emulsion method, nanomotors based on platinum nanoparticles and chitosan chains were synthesized and placed on a substrate of SiO2 nanoparticles using a crosslinking agent containing a disulfide bond. The SiO2 fraction was removed, and the final chitosan-platinum nanomotor was prepared with a bowl-shaped structure. Platinum is the motor of this particle and leads to the movement of nanoparticles in the presence of H2O2 fuel. Therefore, it predicts this nanomotor can move toward the media with higher concentration of peroxide, like cancerous tissues. This nanomotor is designed in such a way that by reaching the media with higher concentration of dithiothreitol (DTT), it stops. This controllability is derived from the change in the morphology of nanomotors from bowl-shape to disc-shape. The results showed that the synthesized nanomotor could act as a biosensor for detecting cancer cells because it is sensitive to specific factors such as pH, H2O2, and DTT. The minimum required values of H2O2 and DTT needed to move the nanomotors were determined by chronoamperometry and found to be 0.1 and 2%, respectively. The release of quercetin as an anti-cancer drug from the nanomotor showed that at acidic pH, the presence of H2O2 and DTT increases the drug release compared to normal conditions. The result of the MTT assay also confirmed that the nanomotors showed more toxicity in the presence of more H2O2 in cancer cells.

Reducing disulfide bonds as a robust strategy to facilitate the self-assembly of cod protein fabricating potential active ingredients-nanocarrier

In this study, a novel method was developed to encapsulate hydrophobic compounds by self-assembly of cod protein (CP) triggered by breaking disulfide bonds. Curcumin (Cur), a representative lipid-soluble polyphenol, was selected as a model to evaluate the potential of CP nanoparticles as novel and accessible nanocarriers. Results showed that the protein structure gradually unfolded with increasing dithiothreitol (DTT) concentration, indicating that S-S cleavage was conducive to forming a looser structure. The resultant unfolded CP exposed more hydrophobic sites, facilitating its interaction with hydrophobic compounds. The encapsulation efficiency (EE) of formed CP-Cur nanoparticles was relatively high, reaching 99.09%, 98.8%, and 89.77% when the mass ratios of CP to Cur were 20:1, 10:1, and 5:1 (w/v), respectively. The hydrophobic interaction, weak van der Waals, and hydrogen bond were the forces contributing to the formation of CP-Cur nanoparticles, whereas the hydrophobic interaction played a crucial role. The CP-Cur complex exhibited increased stability and a homogeneous-stable structural phase. Thus, this research not only proposed a novel and simple encapsulation method of hydrophobic bioactive compounds but also provided a theoretical reference for the application of reductants in food or pharmacy system.

39 The effect of supplementation of different concentrations of dithiothreitol and glutathione on post-thawed semen from Large White boars

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Disulfide bond-breaking induced structural unfolding and assembly of soy protein acting as a nanovehicle for curcumin

Curcumin (Cur), an effective antioxidant agent, suffers a low bioavailability due to its poor solubility and instability in aqueous solutions. With this respect, a facile strategy of encapsulating Cur with unfolded soy protein (SP) through breaking disulfide bonds by dithiothreitol (DTT) was proposed. Multispectral technologies and microstructure showed that the protein structure initially became unfolded and loose in the presence of DTT, then protein nanoparticles were formed, the size of which increased with the increasing DTT concentration. Compared with native SP, SP treated with DTT showed a higher surface hydrophobicity (H0), which might trigger its interaction with Cur to form a soluble compositing nano-architecture. An encapsulation efficiency (EE) higher than 90% was successfully obtained when the mass ratio of Cur to SP was lower than 1:4. Results of fluorescence quenching, Isothermal titration calorimetry (ITC) and Fourier transform infrared spectroscopy (FTIR) further confirmed that the interaction between Cur and SP was mainly driven by hydrophobic interaction, while hydrogen bond and van der Waals force were also involved in. Cur exhibited enhanced thermo-photo stability by forming complex with SP, suggesting its potential utilization in product formulation. Generally, this approach based on reduction technology could be expanded as a facile strategy to boost the solubility and stability of hydrophobic bioactive compounds.

Dithiothreitol-induced reassembly of soybean lipophilic protein as a carrier for resveratrol: Preparation, structural characterization, and functional properties

We employed dithiothreitol (DTT) to reassemble soy lipophilic protein (LP) and increased its solubility for encapsulating resveratrol (Res); we subsequently added hydroxypropyl methylcellulose (HPMC) to further stabilize Res. Physicochemical characterization, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and spectral analysis revealed that DTT triggered the breakage and reassembly of the disulfide bond. Consequently, the solubility of LP increased from 38.64 % to 71.49 %, and the number of free sulfhydryl groups increased to 7.84 mol·g−1. Furthermore, the encapsulation efficiency and structure of reassembled LP nanoparticles loaded with Res were found to be closely related to the DTT concentration used for induction. When HPMC was added, the LP-Res complex demonstrated spontaneous self-assembly, and the pH and temperature stability of the Res in the nanoparticles improved. An in vitro digestion simulation revealed that the reassembled LP was an efficient carrier for Res delivery. Particularly, HPMC improved the bioavailability and sustained release of Res.

Evaluation and minimization of over-alkylation in proteomic sample preparation

Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concentration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.

Use of thiol reagents to disperse cold autoagglutination.

Thiol reagents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) are sulfhydryl reagents that can be used to disperse cold autoagglutinins coating red blood cells (RBCs). DTT and 2-ME are primarily used when warm washing of the coated RBCs fails to successfully disperse the cold autoantibody. Using a weak concentration of DTT or 2-ME, the cold IgM agglutinin can be removed from the coated RBCs without disrupting the IgG or complement coating the RBCs. The treated RBCs can be used for ABO typing, antigen typing, or the direct antiglobulin test.

Dithiothreitol causes toxicity in C. elegans by modulating the methionine-homocysteine cycle.

The redox reagent dithiothreitol (DTT) causes stress in the endoplasmic reticulum (ER) by disrupting its oxidative protein folding environment, which results in the accumulation and misfolding of the newly synthesized proteins. DTT may potentially impact cellular physiology by ER-independent mechanisms; however, such mechanisms remain poorly characterized. Using the nematode model Caenorhabditis elegans, here we show that DTT toxicity is modulated by the bacterial diet. Specifically, the dietary component vitamin B12 alleviates DTT toxicity in a methionine synthase-dependent manner. Using a forward genetic screen, we discover that loss-of-function of R08E5.3, an S-adenosylmethionine (SAM)-dependent methyltransferase, confers DTT resistance. DTT upregulates R08E5.3 expression and modulates the activity of the methionine-homocysteine cycle. Employing genetic and biochemical studies, we establish that DTT toxicity is a result of the depletion of SAM. Finally, we show that a functional IRE-1/XBP-1 unfolded protein response pathway is required to counteract toxicity at high, but not low, DTT concentrations.

Exposure to emamectin benzoate confers cytotoxic effects on human molt-4 T-cells and possible ameliorative role of vitamin E and dithiothreitol

Emamectin benzoate (EMB) is an avermectin insecticide that is extensively used for pest control, but there are few reports concerning its cytotoxic effects on human lymphocytes. In the current study, the hematotoxicity of EMB was evaluated in Molt-4 T-cells, a human T-lymphoblastic cell line with high motility, and the role of vitamin E (VitE) and dithiothreitol (DTT) in attenuating EMB cytotoxicity was characterized. Exposure of Molt-4 cells to EMB decreased cell viability and proliferation, induced a loss of cell clusters, and significantly increased membrane collapse and chromatin condensation. Moreover, EMB significantly increased cell death and suppressed transglutaminase activity. EMB treatment modulated the NF-κB signaling pathway, decreased the expression of p105, p50, and p65/RelA in cytosolic and nuclear fractions, and increased nuclear IκBα expression. EMB increased oxidative stress, as demonstrated by a significant increase in the levels of reactive oxygen species (ROS). Treatment with non-cytotoxic concentrations of VitE or DTT ameliorated the hematotoxicity induced by pretreatment with EMB, increased Molt-4 cell viability, raised the IC50 values of EMB, limited intracellular ROS generation, and mitigated EMB-mediated effects on NF-κB signaling. The results indicate the potential cytotoxicity of EMB on human lymphocytes, and demonstrate that VitE and DTT treatment can reduce the cytotoxic effects of EMB.

Copper complex of a thienyl-hydrazone rhodamine derivative is a highly selective colorimetric sensor for pyrophosphate

Detection of pyrophosphate (PPi) can be used to monitor gene replication, transcription, and other forms of enzymatic DNA/RNA synthesis, but it can be difficult to perform in real samples because of the presence of many other biomolecules and buffer components. In this study we prepared a colorimetric probe that could detect PPi specifically and selectively in the presence of cysteine, dithiothreitol (DTT), nucleoside triphosphates, and human serum (containing several biomolecules and amino acids). Among four candidate rhodamine derivatives, we found that the copper complex 1 could detect PPi uniquely, even in the presence of several competing biomolecules. The copper complex 1 also exhibited its highly selective PPi sensing capability when applied to rolling circle amplification and in vitro transcription in samples containing high concentrations of DTT, making it a promising candidate for colorimetric detection of RNA/DNA when using amplification techniques.

Ligand exchange reactions and PEG stabilization of gold nanoparticles

Gold nanoparticles (Au NPs) were synthesized by the citrate reduction method. Au NPs were aggregated when mixed with mercaptoethanol (ME), dopamine (DAH) and adenine (AD) as the surface passivating citrate molecules were incompletely but heavily replaced by them as supported by the occurrence of a new peak at a longer wavelength and eventual disappearance of the peak/s in the UV–Visible spectra due to the settlement of the aggregated NPs. Transmission electronic microscopic (TEM) images showed aggregated NPs. AD was increasingly replaced by an increasing concentration of dithiothreitol (DTT) in the range of 20 ​μM–10 ​mM, but an incomplete replacement was resulted even after using highly concentrated DTT (10 ​mM). mPEGSH passivates the gold surface where they bind to the gold surface via a mushroom-like configuration. mPEGSH stabilizes the Au NPs preventing aggregation upon mixing with ME, DAH and AD, as revealed by no change in the position and the absorbance of the localized resonance surface plasmon peak in the UV–Visible spectra and well-dispersed Au NPs in the TEM images. Mushroom-like arrangement of mPEGSH on the Au NPs allow adsorption of ME, DAH and AD as revealed by the surface enhanced Raman spectroscopic data.

A continuous spectrophotometric assay for glutathione-independent glyoxalases

We developed a novel continuous assay to quantitatively characterize the catalytic activity of type III methylglyoxalases, a family of enzymes that detoxify methylglyoxal. This assay is based on spectrophotometric detection of hemithioacetal which forms in the reversible reaction of methylglyoxal with dithiothreitol. Due to rapid interconversion between hemithioacetal and methylglyoxal and the known equilibrium constant, hemithioacetal can be quantified spectrophotometrically at 286 nm and used as a reporter for methylglyoxal. When the concentration of methylglyoxal decreases due to catalytic conversion by methylglyoxalases, the concentration of hemithioacetal concomitantly decreases due to its spontaneous decomposition driven by the shift in equilibrium position. Therefore, the rate of total methylglyoxal consumption is the sum of the rate of hemithioacetal decomposition determined spectrophotometrically and the rate of change of methylglyoxal calculated from known concentrations of hemithioacetal. Varying concentrations of dithiothreitol and methylglyoxal creates a broad range of free methylglyoxal in solution that is crucial for the reliable determination of Michaelis constants. We demonstrate the utility of this assay using several recombinant glyoxalases for which kinetic parameters have been determined. This cost-effective and simple assay offers advantages over the existing discontinuous methods and will be useful for quantitative characterization of catalytic activities of type III methylglyoxalases.

Application of hollow mesoporous organosilica nanoparticles as pH and redox double stimuli-responsive nanocontainer in the controlled release of corrosion inhibitor molecules

The stimuli-responsive release could reduce the waste of the corrosion inhibitor and realize the accurate repair of corroded region. In this work, hollow mesoporous organosilica nanoparticles (HMON) were first synthesized by silica-etching chemistry tailoring method. Then, HMON were encapsulated with 2-mercaptobenzothiazole (MBT) corrosion inhibitor molecules to build an intelligent corrosion inhibitor system, i.e., MBT@HMON. The microstructure and frame composition of the synthesized HMON were characterized. After the successful encapsulation of MBT into HMON, the release kinetics of MBT from HMON under different dithiothreitol (DTT) concentration and pH conditions were investigated. Finally, the corrosion inhibition performance of MBT@HMON for copper alloy was evaluated in 3.5 wt% NaCl solution by weight loss test and electrochemical impedance spectroscopy (EIS). The results show that the controlled release of corrosion inhibitor MBT from HMON could be realized by tuning the redox and pH conditions of corrosion medium. When the appropriate concentration of MBT@HMON was added into the corrosion medium (1.6 g·L−1 in this work), the corrosion of cooper alloy was greatly inhibited. This simple and general strategy of introducing corrosion inhibitors loaded HMON directly into the corrosion medium offers a new prospect for the application of nanocontainers in the intelligent corrosion protection of metallic materials.

Aeration mitigates endoplasmic reticulum stress in Saccharomyces cerevisiae even without mitochondrial respiration

Saccharomyces cerevisiae is a facultative anaerobic organism that grows well under both aerobic and hypoxic conditions in media containing abundant fermentable nutrients such as glucose. In order to deeply understand the physiological dependence of S. cerevisiae on aeration, we checked endoplasmic reticulum (ER)-stress status by monitoring the splicing of HAC1 mRNA, which is promoted by the ER stress-sensor protein, Ire1. HAC1-mRNA splicing that was caused by conventional ER-stressing agents, including low concentrations of dithiothreitol (DTT), was more potent in hypoxic cultures than in aerated cultures. Moreover, growth retardation was observed by adding low-dose DTT into hypoxic cultures of ire1Δ cells. Unexpectedly, aeration mitigated ER stress and DTT-induced impairment of ER oxidative protein folding even when mitochondrial respiration was halted by the ρo mutation. An ER-located protein Ero1 is known to directly consume molecular oxygen to initiate the ER protein oxidation cascade, which promotes oxidative protein folding of ER client proteins. Our further study using ero1-mutant strains suggested that, in addition to mitochondrial respiration, this Ero1-medaited reaction contributes to mitigation of ER stress by molecular oxygen. Taken together, here we demonstrate a scenario in which aeration acts beneficially on S. cerevisiae cells even under fermentative conditions.

Optimization of a fluorogenic assay to determine caspase 3/7 activity in meat extracts.

Usefulness of general-purpose fluorogenic assay kits to determine caspase 3/7 activity of biological extracts is highly compromised in meat-based samples due to their scarce enzyme concentration. In the present work, a straightforward protocol is presented with two main purposes: 1) to enhance sensitivity of the fluorogenic approach addressing caspase 3/7 activity in tissues showing scarce enzyme concentration such as skeletal muscle, and 2) to reduce/economize the volume of employed reagents. The enzyme extraction procedure, peptide substrate, dithiothreitol concentration and detection settings were appropriately optimized for use in microtiter-plate fluorometers. As a result, low to high enzyme activity extracts (from 10,000 to 260,000 relative fluorescence units) can be measured under developed sampling and experimental conditions. The fact that enzyme reactions took place in 96-microtiter well plates reduces the consumption of chemical compounds when analysing a high number of samples, thus contributing to environment sustainability.

Reduction and temperature-responsive hydrogel composed of hydroxyethyl disulfide-bis-glycidyl ether-crosslinked poly(hydroxyethyl acrylate-co-methyl methacrylate)

Reduction and temperature-responsive gels were prepared by crosslinking poly(hydroxyethyl acrylate-co-methyl methacrylate) (poly(HEA-co-MMA)) using hydroxyethyl disulfide-bis-glycidyl ether(HEDSGE) as a disulfide cross-linker. Poly(HEA-co-MMA) exhibited lower critical solution temperature (LCST) behavior when MMA content was 7.4 and 8.3 mole %. Energy dispersive X-ray and Raman spectroscopy revealed that the copolymer chains of the gels were cross-linked by disulfide bond (i.e., HEDSGE). The gels released their content in proportion to the concentration of a reducing agent (i.e., dithiothreitol). In addition, the gels released their payloads in a temperature dependent manner, possibly due to the LCST property of the copolymer. Highlights Reduction and thermo-responsive gels were prepared by crosslinking poly(HEA-co-MMA) using HEDSGE. Poly(HEA-co-MMA) exhibited LCST behavior when MMA content was 7.4 mole % and 8.3 mole %. Gels released their content in proportion to dithiothreitol (a reducing agent) concentration. Gels released their payloads in a temperature dependent manner.