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A key challenge in developing Machine Learning (ML) models for predicting or diagnosing Cardiovascular Disease (CVD), is selecting suitable algorithms and fine-tuning their parameters. In this study, we employed three ML techniques, namely Auto-WEKA, Decision Table/Naive Bayes (DTNB), and Multiobjective Evolutionary (MOE) fuzzy classifier to create diagnostic models using the Heart Disease Dataset from IEEE Dataport. Auto-WEKA generated a highly accurate model with a 100% success rate through optimal classifier selection and hyperparameter configuration. The DTNB classifier yielded a satisfactory 85.63% prediction accuracy concerning patients' risk levels. Further refinements, though, could help reduce possible misclassifications. Finally, the MOE fuzzy classifier achieved approximately 81.6% accuracy, indicating the potential for enhancing precision and recall values by adjusting classifier settings. Our findings underscore the promise of ML tools in CVD diagnosis and suggest further optimization of classifier parameters for superior performance. [ABSTRACT FROM AUTHOR]

ABSTRACTMicrobial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell–cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial–neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4–15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5′-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial–neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy. [ABSTRACT FROM AUTHOR]

Paclitaxel (PTX) is frequently utilized for the chemotherapy of breast cancer, but its continuous treatment provokes hyposensitivity. Here, we established a PTX-resistant variant of human breast cancer MCF7 cells and found that acquiring the chemoresistance elicits a remarkable up-regulation of aldo-keto reductase (AKR) 1C3. MCF7 cell sensitivity to PTX toxicity was increased by pretreatment with AKR1C3 inhibitor and knockdown of this enzyme, and decreased by its overexpression, inferring a crucial role of AKR1C3 in the development of PTX resistance. The PTX-resistant cells were much less sensitive to 4-hydroxy-2-nonenal and acrolein, cytotoxic reactive aldehydes derived from ROS-mediated lipid peroxidation, compared with the parental cells. Additionally, the resistant cells lowered levels of 4-hydroxy-2-nonenal formed during PTX treatment, which was mitigated by pretreating with AKR1C3 inhibitor, suggesting that AKR1C3 procures the chemoresistance through facilitating the metabolism of the cytotoxic aldehyde. The gain of PTX resistance additively promoted the aberrant expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with AKR1C3 and ABCB1 inhibitors overcame the PTX resistance and cross-resistance to another taxane-based drug docetaxel. Collectively, combined treatment with AKR1C3 and ABCB1 inhibitors may exert an overcoming effect of PTX resistance in breast cancer. [ABSTRACT FROM AUTHOR]

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell–cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5′-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial–neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis. [ABSTRACT FROM AUTHOR]

In SARS-CoV-2-infected humans, disease progression is often associated with acute respiratory distress syndrome involving severe lung injury, coagulopathy, and thrombosis of the alveolar capillaries. The pathogenesis of these pulmonary complications in COVID-19 patients has not been elucidated. Autopsy study of these patients showed SARS-CoV-2 virions in pulmonary vessels and sequestrated leukocytes infiltrates associated with endotheliopathy and microvascular thrombosis. Since SARS-CoV-2 enters and infects target cells by binding its spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2), and there is evidence that vascular endothelial cells and neutrophils express ACE2, we investigated the effect of S-proteins and cell–cell communication on primary human lung microvascular endothelial cells (HLMEC) and neutrophils expression of thrombogenic factors and the potential mechanisms. Using S-proteins of two different SARS-CoV-2 variants (Wuhan and Delta), we demonstrate that exposure of HLMEC or neutrophils to S-proteins, co-culture of HLMEC exposed to S-proteins with non-exposed neutrophils, or co-culture of neutrophils exposed to S-proteins with non-exposed HLMEC induced transcriptional upregulation of tissue factor (TF), significantly increased the expression and secretion of factor (F)-V, thrombin, and fibrinogen and inhibited tissue factor pathway inhibitor (TFPI), the primary regulator of the extrinsic pathway of blood coagulation, in both cell types. Recombinant (r)TFPI and a thiol blocker (5,5′-dithio-bis-(2-nitrobenzoic acid)) prevented S-protein-induced expression and secretion of Factor-V, thrombin, and fibrinogen. Thrombomodulin blocked S-protein-induced expression and secretion of fibrinogen but had no effect on S-protein-induced expression of Factor-V or thrombin. These results suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial–neutrophil interactions, viral S-proteins induce coagulopathy via the TF pathway and mechanisms involving functional thiol groups. These findings suggest that using rTFPI and/or thiol-based drugs could be a viable therapeutic strategy against SARS-CoV-2-induced coagulopathy and thrombosis.

In aqueous acetate buffer media (pH=4.0-5.2), reaction between the superoxo complex, [(dien)(en)CoIII(O2)CoIII (en) (dien)]5+ (1) and pyridine N-oxide (C5H5NO) proceeds through a complex kinetic pathway and the change of the absorbance of 1 with time also follows a complex pattern. Only in the presence of Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB), the reaction becomes quantitative and each mole of 1 is reduced by one mole of pyridine Noxide (C5H5NO) to produce the corresponding hydroperoxo complex, [(dien)(en)CoIII (HO2)CoIII(en)(dien)]5+ (2), following proton coupled electron transfer (PCET) path. In presence of DTNB, with large excess of pyridine N-oxide (C5H5NO) over [1], the reaction follows first-order kinetics and the observed rate constant, ko increase linearly with increase in [C5H5NO] but decreases with increase in [H+]. It is also noticed that the reaction rate significantly decreases with increasing proportion of D2O replacing H2O in the solvent. Therefore, an H-atom transfer (HAT) from the reducing species to the bridging superoxide in 1 seems reasonable at the rate determining step. Indeed the present work confesses the forthcoming involvement of Ellman's reagent as a radical scavenger. [ABSTRACT FROM AUTHOR]

Discovery of surface‐enhanced Raman scattering (SERS) followed by evolution of optical systems and nanoengineering approaches has paved a path to detection of essential organic molecules on solid SERS‐active substrates from solutions at concentrations attributed to single‐molecule ones, i. e. below 10−15 M. However, in practical terms confident SERS‐imaging of single molecules is still quite a challenge. In present work, we fabricated and comprehensively characterized tightly‐packed 3D silver dendrites with prevalent chevron morphology that demonstrated ultrahigh sensitivity as SERS‐active substrates resulted in 10−18 M detection limit. Using these substrates we achieved SERS‐imaging of single 5‐thio‐2‐nitrobenzoic acid (TNB) molecule released from the attomolar‐concentrated solution of of 5,5′‐dithio‐bis‐[2‐nitrobenzoic acid] (DTNB), which is vital compound for chemical and biomedical analysis. In contrast to generally accepted belief about adsorption of only uniform monomolecular TNB layer on surface of silver nanostructures, we showed formation of a coating constituted by TNB layer and DTNB nanoclusters on the dendrites' surface at 10−6–10−12 M DTNB concentrations confirmed by presence/absence of disulfide bonds signature in the SERS‐spectra and by scanning electron microscopy. DTNB concentrations below 10−14 M resulted in adsorption of TNB molecules in separated spots on the surface of silver nanostructures. [ABSTRACT FROM AUTHOR]

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c -type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling. IMPORTANCE Organic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c -type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems., Competing Interests: The authors declare no conflict of interest.

Subclinical mastitis is one of the most important infectious diseases, and higher levels of oxidative stress in the peritpartum period were related to higher risk of mastitis. The objective of the present study was to evaluate the diagnostic value of milk oxidative stress variables in early lactation of dairy cow with subclinical mastitis. A total of 925 milk samples from quarters of cows during (7 ± 3) days post-calving from two dairy herds were included in the present study. Bacterial culture, somatic cell count (SCC), DTNB (dithiobis nitrobenzoic acid for oxidative capacity), and FRAP (ferric reducing antioxidant power for antioxidatant capacity) have been assayed. Receiver-operating characteristic (ROC) analysis was used to assess the accuracy diagnostic value of each test using bacterial culture as gold standard. The accuracy of DTNB assay was comparable to that observed for SCC (AUC = 0.724 vs. AUC = 0.739). Sensitivity and specificity of DTNB at the concentration of 2.00 μmol s−1 were also comparable to those for SCC at 165 × 103 SCCml−1 (59.1 and 80.7% vs. 56.9 and 82.2%, respectively). Concentrations of both DTNB and FRAP were significantly higher in milk samples of quarters infected with S. aureus, when compared to non-infected quarters and quarters infected with other bacteria. The results showed that colorimetric measurement of DTNB could be a valuable indicator of subclinical mastitis, especially infection caused by S. aureus. [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thioldisulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N-ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membranebound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. [ABSTRACT FROM AUTHOR]

Hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferases are BAHD family acyltransferases that transfer hydroxycinnamoyl moieties from a CoA-thioester to an acceptor amine or alcohol to form an N -hydroxycinnamoyl amide or O -hydroxycinnamoyl ester, respectively, with the concomitant release of free CoA. One approach to measure reaction rates for these enzymes is to quantify the hydroxycinnamoyl amide or ester reaction product following chromatographic separation of reaction components. This approach can be labor-intensive and time-consuming. As an alternative, we examined the use of 5,5′-dithio- bis -(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) to spectrophotometrically quantify, in real time, the release of free CoA during the transferase reaction. Using a hydroxycinnamoyl-CoA: l -DOPA hydroxycinnamoyl transferase as a model, we show that DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters. This approach should be applicable to a wide range of hydroxycinnamoyl-CoA and other BAHD acyltransferases. [ABSTRACT FROM AUTHOR]

In this work, a simple, fast and sensitive fluorescent sensor based on N-doped carbon quantum dots (NCQDs) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was fabricated for selective detection of biothiols. NCQDs were successfully prepared via hydrothermal method and characterized using TEM, XRD, Raman spectrometer, FT-IR and XPS. In the presence of biothiols in the mixture of NCQDs and DTNB, −SH of biothiols can interact with DTNB and subsequently affording yellow-colored 5-thio-2-nitrobenzoate (TNB), which can strongly absorb excitation light of NCQDs and lead to fluorescence quenching of NCQDs. As a consequence, quantitative screening of biothiols can be achieved. Under optimized conditions, good linear relationships in the range of 0.05–40 μM was obtained for L -cysteine (L-Cys), 0.01–40 μM for glutathione (GSH), and 0.05–40 μM for homocysteine (HCys). The detection limits of this assay for L-Cys, GSH and HCys were 1.01 nM, 3.23 nM, and 3.29 nM, respectively. And selectivity, reproducibility and stability of the sensor were also evaluated. These results demonstrated that the presented fluorescent sensor can be a promising sensing platform for biothiols analysis. [ABSTRACT FROM AUTHOR]

Introduction Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl- l -cysteine (NAC) or glutathione (GSH). Methods BEAS-2B cells were exposed for 24 h to CSE, H 2 O 2 , acrolein, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63 + CD81 + bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. Results CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H 2 O 2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. Summary/conclusion Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. [ABSTRACT FROM AUTHOR]

An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 μM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time. [ABSTRACT FROM AUTHOR]

Purpose: Coffee is a widely consumed beverage. While recent studies have linked its intake to a reduced risk of cataracts, caffeine is believed to be the key factor for its effect. To know how roasting beans affects the effect of coffee on cataract formation, we investigated the impact roasting using a selenite-induced cataract rat model. Materials and Methods: Sprague Dawley rats were given a single injection of sodium selenite, which induced formation of nuclear cataracts by day 6, with or without coffee intake (100% coffee, 0.2 mL/day) for following 3 days. Results: The concentrations of glutathione (GSH) and ascorbic acid (AsA) in selenite-induced cataract lenses declined to half that of controls. However, 3 days of coffee intake ameliorated the reduction of GSH and AsA so that concentrations remained at 70–80% that of controls. Roasting enhanced the preventive effect of coffee by further reducing cataract formation and ameliorating selenite-induced reduction of antioxidants. High-performance liquid chromatography analysis revealed degradation of chlorogenic acid and generation of pyrocatechol during the coffee roasting process. We discovered that pyrocatechol, at doses equivalent to that found in dark-roasted coffee, was equally effective as caffeine at reducing cataract formation and ameliorating the reduction of antioxidants. Conclusion: Our results indicate that pyrocatechol, generated during the roasting process, acts as an antioxidant together with caffeine to prevent cataract formation. [ABSTRACT FROM PUBLISHER]

In aqueous acetate buffer, reaction between the superoxo complex [(dien)(en)CoIII(O2)CoIII(en)(dien)]5+(1) and thiourea (Tu, SC(NH2)2) proceeds through a complex kinetic pathway and the change of the absorbance of1with time follows a sigmoidal trace. Only in the presence of Ellman’s reagent (5,5′-dithio-bis-(2-nitrobenzoic acid), DTNB), the reaction becomes quantitative and Tu reduces the bound superoxo group (––) of1to the corresponding peroxo group (––) in [(dien)(en)CoIII(O2)CoIII(en)(dien)]4+(2). In the presence of DTNB, with large excess of Tu over [1], the reaction follows first-order kinetics and the observed rate constant,ko, increases linearly with the increase in [1], [TTu] ([TTu] is the analytical concentration of Tu), [H+], and the media ionic strength (I). The presence of an intercept in the plot ofkoversus[H+] suggests that both tautomeric forms of thiourea, i.e. TuZand its protonated form TuZH+, are kinetically reactive reductants. The calculated rate constant values fork1(~102) andk2(~104) indicate that TuZH+plays the leading role in the redox reaction. The present work also reveals the prospective involvement of Ellman’s reagent as a radical scavenger. [ABSTRACT FROM AUTHOR]

This commentary features the groundbreaking manuscript published in the 1959 issue of Archives of Biochemistry and Biophysics by George L. Ellman. The studies describe the quantification of thiols in tissues and purified proteins using DTNB (Ellman's Reagent). This highly referenced manuscript is recognized in this anniversary issue because of the impact these studies have played across diverse scientific fields. [ABSTRACT FROM AUTHOR]

BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near-real time measurement of reaction rates, permitting reaction conditions (buffer, reactant, and enzyme concentrations, etc.) to be changed "on the fly." The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase, a BAHD acyl-CoA hydroxycinnamoyltransferase not previously characterized with respect to kinetic parameters. This approach may be more generally applicable to transferases using CoA donors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

β-Lactoglobulin (β-Lg) is relatively unstable during thermal processes used in the dairy industry. One of many ways to improve the heat stability of β-Lg is by chemical modification by protein–reagent binding. Potential reagents, such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM), bind to the exposed free sulfhydryl (–SH) group of denatured β-Lg and prevent it from being involved in protein aggregation reactions. However, no study has shown the specific binding of these reagents to β-Lg. Furthermore, the question of how many molecules of these reagents bind to β-Lg remains unanswered. In this research, the reactions of DTNB or NEM with the reactive monomers of β-Lg were identified by the presence of bands for 5-thio-2-nitrobenzoyl (TNB)- or NEM-modified monomers on Native-PAGE and Non-Reducing (NR) sodium deodecyl sulphate (SDS)-PAGE gels, and by an additional mass of β-Lg monomers corresponding to a molecule of TNB or NEM in mass spectra. [ABSTRACT FROM AUTHOR]

In this study, we developed the genetically modified organism detection method by using the combination of rolling circle amplification (RCA) and surface-enhanced Raman spectroscopy (SERS). An oligonucleotide probe which is specific for 35S DNA promoter target was immobilised onto the gold slide and a RCA reaction was performed. A self-assembled monolayer was formed on gold nanorods using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and the second probe of the 35S DNA promoter target was immobilised on the activated gold coated slide surfaces. Probes on the nanoparticles were hybridised with the target oligonucleotide. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. SERS spectra of target molecules were enhanced through the RCA reaction and the detection limit was found to be 6.3 fM. The sensitivity of the developed RCA–SERS method was compared with another method which had been performed without using RCA reaction, and the detection limit was found to be 0.1 pM. The correlation between the target concentration and the SERS signal was found to be linear, within the range of 1 pM to 10 nM for the traditional assay and 100 fM to 100 nM for the RCA assay. For the developed RCA–SERS assay, the specificity tests were performed using the 35S promoter of Bt-176 maize gene. It was found out that the developed RCA–SERS sandwich assay method is quite sensitive, selective and specific for target sequences in model and real systems. [ABSTRACT FROM AUTHOR]

Heteropterans produce 2-alkenals and 4-keto-2-alkenals that function as defense substances or pheromones. However, in spite of advances in heteropteran chemistry, it is still unclear how these compounds affect insect physiology. We found that exposure to 4-oxo-( E )-2-hexenal (OHE) induced permanent paralysis and death in crickets, an experimental model. The depletion of free thiols in leg tissues of OHE-treated crickets and the in vitro adduct formation of OHE with a thiol compound suggest that covalent binding of OHE to biologically active thiols is a potential cause affecting crickets’ locomotion. [ABSTRACT FROM AUTHOR]