Tris 2-carboxyethyl Phosphine Research Articles

Establishment of disulfidptosis-related LncRNA signature as biomarkers in colon adenocarcinoma.

Metabolic reprogramming is a hallmark of cancer and plays a key role in precision oncology treatment. Long non-coding RNAs (lncRNAs) regulate cancer cell behavior, including metabolism. Disulfidptosis, a newly identified form of regulated cell death triggered by glucose starvation, has yet to be fully understood in colon adenocarcinoma (COAD). This study aimed to confirm the existence and role of disulfidptosis in COAD and identify disulfidptosis-related lncRNAs that may be targeted to induce disulfidptosis in COAD. PI and F-actin staining were used to observe disulfidptosis in COAD cell lines. Disulfidptosis-related lncRNAs were identified based on the expression of disulfidptosis-associated genes in the TCGA-COAD database. A four-lncRNA signature for disulfidptosis was established. Subsequently, loss-of-function assays explored the roles of AC013652.1 and MCM3AP-AS1 in disulfidptosis. Disulfidptosis was observed in COAD cells under glucose starvation and could be reversed by agents that prevent disulfide stress, such as dithiothreitol (DTT) and tris-(2-carboxyethyl)-phosphine (TCEP). The prognostic value of disulfidptosis-associated genes in COAD patients was confirmed, with higher expression indicating longer survival. A disulfidptosis-related lncRNA signature comprising four lncRNAs was established based on the expression of these genes. Among these, AC013652.1 and MCM3AP-AS1 predicted worse prognoses. Furthermore, inhibiting AC013652.1 or MCM3AP-AS1 increased disulfidptosis-associated gene expression and cellular death, which could be reversed by DTT and TCEP. This study provides hitherto undocumented evidence of the existence of disulfidptosis and the prognostic value of disulfidptosis-associated genes in COAD. Importantly, we identified lncRNAs AC013652.1 and MCM3AP-AS1, which suppress disulfidptosis and may serve as potential therapeutic targets for COAD.

Biomimetic Remineralization of Dental Hard Tissues via Amyloid‐Like Protein Matrix Composite with Amorphous Calcium Phosphate

AbstractNumerous remineralizing coatings aim to prevent or treat early enamel lesions and occlude exposed dentinal tubules (DTs). Nevertheless, the pace of remineralization is inadequate, and the mechanical robustness of the newly established mineral layer fails to match the inherent strength. In this study, a biomimetic mineralization strategy aimed at replicating key events in biological mineralization, specifically focusing on the organic–inorganic composite matrix, is proposed. The material utilizes Tris(2‐carboxyethyl)phosphine (TCEP), which serves a dual role: stabilized amorphous calcium phosphate (ACP) (ACP@TCEP) nanoparticles as its inorganic component, and catalyzing the cleavage of intramolecular disulfide bonds in poly(ethylene glycol) (PEG) grafted lysozyme (lyso‐PEG) to facilitate the formation of an amyloid‐like protein matrix composite with ACP (ACP@lyso‐PEG nanocomplexes). ACP@lyso‐PEG nanocomplexes can rapidly and efficiently form an enamel‐like remineralization layer on the surface of damaged dental hard tissue, reaching ≈4.205 µm thickness after 3 days of acid‐etched enamel. Furthermore, achieving a depth of DTs occlusion exceeding 60 µm after 5 days, using a simple immersion process. The resulting mineralized layer exhibits mechanical strength comparable to natural teeth. This study introduces a conceptual biomimetic mineralization strategy for effective enamel repair or DTs occlusion in clinical practices, and offers potential insights into the mechanisms of biomineral formation.

Pollution characteristics and risk assessment of organophosphate esters (OPEs) in typical industrial parks in Southwest China.

As alternative substances of PBDEs, organophosphate esters (OPEs), an emerging organic pollutant, were increasingly produced and used in many kinds of industries and consumer products. However, OPEs also have various adverse toxic effects. Information on the pollution levels and exposure to OPEs in related industries is still limited. This study presented data on OPE contamination in the soil, leaf, and river water samples from seven typical industrial parks in Southwest China. Total concentration of seven OPEs (Σ7OPE) including tri-n-butyl phosphate (TnBP), tris-(2-ethylhexyl) phosphate (TEHP), tris-(2-butoxyethyl) phosphate (TBEP), tris-(2-carboxyethyl) phosphine (TCEP), triphenyl phosphate (TPhP), tris-(1,3-dichloro-2-propyl) ester (TDCPP), and tris-(chlorisopropyl) phosphate (TCPP) in the soil samples (36.2 ~ 219.7 ng/g) and the surrounding river water samples (118.9 ~ 287.7 ng/L) were mostly lower than those in other studies, while the Σ7OPE level in the leaves (2053.3 ~ 8152.7 ng/g) was relatively high. There were significant differences in the concentration and distribution of OPEs in the surrounding environment of different industrial parks. TDCPP, TnBP, and TCPP could be used as the characteristic compound in soil samples from auto industrial park, river samples from shoe making industrial park, and leaf samples from logistics park, respectively. The parameter m (the content ratio of chlorinated OPEs to alkyl OPEs) was suggested to distinguish the types of industrial park preliminary. When m ≥ 1, it mainly refers to heavy industries sources such as automobiles, electronics, and machinery, etc. When m<1, it mainly for the light industrial sources such as textile industry, transportation services, and resources processing, etc. For logistics park, furniture park and Wuhou comprehensive industrial park, the volatilization of materials was the main sources of OPEs in the surrounding environment, while more effort was required to strengthen the pollution control and management of the waste water and soil in the pharmacy industrial park, shoe making industrial park and auto industrial park. Risk assessment showed that there was a negligible non-cancer and carcinogenic risk in the soil, while high attention should be paid to the non-cancer risk for children.

Acetaminophen used at therapeutic dosage causes significant changes in signaling pathways in mouse hearts

Acetaminophen (APAP) is a common over-the-counter medication that is used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses for a short amount of time. However, recent research suggests that APAP taken regularly can result in increased blood pressure, resulting in increased cardiovascular risk. Previous publications found that non-steroidal anti-inflammatory drugs (NSAIDs) resulted in increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not an NSAID, it is likely that APAP has similar effects. Our hypothesis is that regular usage of APAP increases ROS levels, leading to potential dysfunction in hearts and other organs. We treated five 6-month-old female mice with either water or acetaminophen dissolved in water for seven days. The concentration of acetaminophen that mice were given was 103 mg/kg/day, which is equivalent to a daily 500 mg dosage of acetaminophen in humans. The heart samples from these mice were homogenized in urea lysis buffer and then centrifuged to separate the supernatant from the pellet. After discarding the pellet, the proteins in the supernatant were discarded, and the pellet was treated with acetone overnight to allow the protein to precipitate. The supernatant was discarded, and the pellet was kept and denatured with tris(2-carboxyethyl) phosphine (TCEP) while incubating in a 55ºC dry bath, iodoacetamide was added to prevent disulfide bonds from forming, and acetone was added to the supernatant and left overnight to make proteins precipitate. After precipitation, the sample was centrifuged again to separate the supernatant from the pellet, and the pellet was saved while the supernatant was discarded. The pellet was resuspended and Tandem Mass Tags (TMT) label reagents were added to the samples, the samples quenched, and then combined and analyzed using mass spectroscopy (MS) after fractionating the samples using a Pierce High pH Reverse-Phase Peptide Fractionation kit (Thermofisher), then separated and eluted in liquid chromatography (LC) to determine any changes in the protein. LC/MS/MS data underwent a power analysis and Sequest-HT to compare samples to all mouse sequences. Target Decoy PSM was used to analyze and exclude decoy sequences from the samples, and Scaffold Q+ was used to quantify the peptides and proteins identified from the MS and LC data. Pathway analysis of the significant protein expression changes suggests antioxidant pathways, the ubiquitin-proteasome system, fatty acid oxidation, oxidative phosphorylation, and de novo purine synthesis and degradation. Proteasome assays were also conducted for the β5, β2, and β1 proteolytic subunits. There was a significant decrease in the β5 chymotrypsin-like activity in the hearts of APAP-treated mice compared to the control mice and no significant change in the β2 and β1 proteasome activity. Overall, these results show that mice treated with a therapeutic dosage of APAP regularly over the course of a week exhibited increased stress in the heart. NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Accurate measurement of sulfhydryls and TCEP-releasable sulfhydryls in the liquid phase of wine that contribute to ‘reductive’ aromas using LC-MS/MS

Volatile sulfur compounds (VSCs) are important aroma and flavour characters in food and beverage products. The identification and quantification of these extremely reactive and volatile compounds pose analytical challenges which demand selective and sensitive methods. In this study, a novel quantification method was developed to analyse sulfhydryls as well as the total pool of sulfhydryls which can be released after tris(2-carboxyethyl)phosphine (TCEP) addition from disulfides, polysulfides, metal-bound and other yet to be identified sources naturally present in wine. The majority of methods for VSC quantification analyse VSCs in wine headspace, whereas this method measures sulfhydryls and TCEP-releasable sulfhydryl species, which likely include free and metal-bound sulfhydryl forms, in the liquid phase of wine using UHPLC-MS/MS. Sulfhydryls were derivatised with N-(2-ferroceneethyl) maleimide (FEM), subsequently, followed by differential labelling of sulfhydryls released after TCEP addition with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA). Analysis of commercial wines revealed the presence of hydrogen sulfide, methanethiol, ethanethiol, and 2-mercaptoethanol at aroma-active concentrations. Significant positive correlations were found between MeSH and CH3–S-R TCEP-releasable species, and significant positive correlations were found between EtSH and CH3–CH2–S-R TCEP-releasable species. This method provides important information on sulfhydryls, and may also provide insights into a wine's risk of developing ‘reductive’ faults post-bottling from latent sources.

Diagonal Chromatography for the Isolation of O-Linked β-N-acetyl-D-Glucosaminylated (O-GlcNAcylated) Peptides in HeLa Cell Extracts

Protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification process that plays an essential role in biological activities. Growing evidence indicates that aberration of O-GlcNAcylation is associated with various diseases, e.g. diabetes, neurological diseases, and cancers. However, the mechanistic studies of O-GlcNAcylation are lagging behind other post-translational modifications due to its extremely low abundance, limited analytical tools, and specificity. Herein, diagonal strong cation exchange chromatography was applied to enrich the O-GlcNAc glycosylated peptides prior to mass spectrometric analysis by liquid chromatography/ion trap tandem mass spectrometry (LC–MS/MS). In this strategy, the O-GlcNAcylated peptides were first enzymatically labeled with an azide-modified galactosamine (GalNAz) and fractionated by strong cation exchange (SCX) chromatography. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in GalNAz-modified peptides to a primary amine group. TCEP-induced reduction of GalNAz-modified peptides was separated from unmodified peptides by diagonal SCX. By reversed-phase LC–MS/MS analysis of secondary SCX fractions, O-GlcNAcylated peptides were isolated and identified from the mixtures of O-GlcNAc-modified and unmodified peptides in HeLa cell extract. A total of 250 O-GlcNAcylation sites on 215 proteins were identified. Therefore, this novel method could be a potential tool for the isolation and site analysis of O-GlcNAc-modified peptides.

Bovine Serum Albumin/Polyvinyl Alcohol Double‐Network Hydrogel Containing ϵ‐Polylysine for Antibacterial Performance

AbstractWith the discovery and abuse of antibiotics, bacterial resistance has become a key problem in the field of antibacterial materials. Therefore, antibacterial materials that can effectively inhibit bacterial infection are considered to be important biological materials. In this study, a double‐network hydrogel system was designed using bovine serum albumin (BSA) and polyvinyl alcohol (PVA) as the main body. The first network was formed by reducing the disulfide bond on BSA to sulfhydryl group by tris (2‐carboxyethyl) phosphine (TCEP). The second network of PVA was used as the hydrogel to prepare a double‐network hydrogel with good mechanical strength. The introduction of ϵ‐polylysine gives the hydrogel antibacterial activity, which enables it to destroy the cell structure and kill bacteria through electrostatic interaction, and can effectively solve the problem of drug resistance in the field of antibacterial medicine.

Dual Barrier‐Penetrating Inhaled Nanoparticles for Enhanced Idiopathic Pulmonary Fibrosis Therapy

AbstractIdiopathic pulmonary fibrosis (IPF) is an age‐related pulmonary interstitial disease with unclear etiology that poses a serious threat to human health. IPF interventions in clinical settings mainly involve oral medications, such as nintedanib (NIN), which exhibit limited accumulation in the lungs and neglect the epithelial micro‐environment. Inhalation is an efficient route for the treatment of pulmonary diseases. However, the mucus barrier in the trachea and the extracellular matrix (ECM) barrier in the interstitium are the two main obstacles to inhaled therapeutic agent delivery. Therefore, in this study, dual barrier‐penetrating inhaled liposomes (AN‐TR) are constructed utilizing tris‐(2‐carboxyethyl)‐phosphine (TCEP) and l‐arginine to penetrate the mucus and ECM barriers, respectively. This approach facilitates the thorough and uniform distribution of NIN and navitoclax (ABT‐263) across all five lung lobes. Furthermore, ABT‐263 can remove the senescent epithelial cells in the trachea and alveoli, thereby improving the efficiency of NIN for IPF treatment. This study suggests dual barrier‐penetrating inhaled liposomes as efficient noninvasive vehicles for first‐line clinical medications to improve the efficacy of IPF treatment.

Optimization of hybrid gelatin-polysaccharide bioinks exploiting thiol-norbornene chemistry using a reducing additive

Thiol-norbornene chemistry offers great potential in the field of hydrogel development, given its step growth crosslinking mechanism. However, limitations exist with regard to deposition-based bioprinting of thiol-containing hydrogels, associated with premature crosslinking of thiolated (bio)polymers resulting from disulfide formation in the presence of oxygen. More specifically, disulfide formation can result in an increase in viscosity thereby impeding the printing process. In the present work, hydrogels constituting norbornene-modified dextran (DexNB) combined with thiolated gelatin (GelSH) are selected as case study to explore the potential of incorporating the reducing agent tris(2-carboxyethyl)phosphine (TCEP), to prevent the formation of disulfides. We observed that, in addition to preventing disulfide formation, TCEP also contributed to premature, spontaneous thiol-norbornene crosslinking without the use of UV light as evidenced via 1H-NMR spectroscopy. Herein, an optimal concentration of 25 mol% TCEP with respect to the amount of thiols was found, thereby limiting auto-gelation by both minimizing disulfide formation and spontaneous thiol-norbornene reaction. This concentration results in a constant viscosity during at least 24 h, a more homogeneous network being formed as evidenced using atomic force microscopy while retaining bioink biocompatibility as evidenced by a cell viability of human foreskin fibroblasts exceeding 70% according to ISO 10993-6:2016.

High-Density Immobilization of TCEP on Silica Beads for Efficient Disulfide Reduction and Thiol Alkylation in Peptides.

Tris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15 μmol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129±16 μmol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.

Bottom-Up Extrusion-Based Biofabrication of the Osteoid Niche.

Bone regeneration remains a clinical challenge given the transplantation incidence rate and the associated economic burden. Bottom-up osteoid tissue engineering has the potential to offer an alternative approach to current clinical solutions that suffer from various drawbacks. In this paper, deposition-based bioprinting is exploited while the effect is explored of both the crosslinking mechanism (gelatin methacryloyl (GelMA) versus gelatin norbornene (DS 91) crosslinked with thiolated gelatin (GelNBSH)) and the degree of substitution (GelNBSH versus norbornene-norbornene-modified gelatin (DS 169) crosslinked with thiolated gelatin (GelNBNBSH)) on the presented biophysical cues as well as on the osteogenic differentiation. The incorporation of tris(2-carboxyethyl)phosphine (TCEP) to the step-growth inks allows the production of reproducible and biocompatible scaffolds based on thiol-ene chemistry. Dental pulp stem cell encapsulation in GelNBNBSH biofabricated constructs shows a favorable response due to the combination of its stress relaxation and substrate rigidity (bulk compressive modulus of 11-30kPa) as reflected by a sevenfold increase in calcium production compared to the tissue engineering standard GelMA. This work is the first to exploit a controlled biocompatible and cell-interactive thiolated macromolecular crosslinker (GelSH + TCEP) allowing the extrusion-based biofabrication of low concentration (5 w/v%) modified osteogenic gelatin-based inks (GelNBNBSH + TCEP).

Development of Next Generation Antibody-Polymer-Drug Conjugates for Treatment of Multiple Myeloma

Background: Antibody-drug conjugates (ADCs) are an effective class of cancer therapeutics, but current ADCs face stability challenges, resulting in premature payload release and off-target toxicity. To overcome this, we developed a novel strategy using antibody-polymer-based drug conjugates (pADCs). Our pADCs have a number of advantages compared to currently FDA approved ADCs, including: 1) Increased drug payload without altering binding site numbers, ensuring maximum drug loading without affecting antigen and Fc binding; 2) Potential for synergistic immunotherapy and chemotherapy; 3) Improved pharmacokinetics and reduced immunogenicity with modification using water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, providing significant stealth effect; and 4) Enhanced stability under physiological conditions, reducing premature payload release and off-target toxicity. Panobinostat, a pan-HDAC (histone deacetylase) inhibitor, enhances CD38 expression and improves pADC efficiency against MM cells. Our hypothesis is that our pADC system induces strong cytotoxicity, further enhanced by panobinostat-induced CD38 upregulation, leading to more efficient treatment outcomes. Methods:mAbs decorated with cyclooctyne (DBCO) moieties in the hinge region were prepared in two steps: mAbs were reduced with tris(2-carboxyethyl)phosphine (TCEP), then thiol groups were capped with DBCO-PEG4-Mal. Azide-functionalized semitelechelic (ST) polymer-drug conjugates (ST-P-drug-N3) were synthesized via RAFT polymerization. Antibody-polymer-drug conjugates were prepared by CLICK chemistry. MM.1S and RPMI 8226 cell lines (ATCC) were used as the MM cell model. The cell internalization was revealed by confocal imaging. Primary MM cells were collected from the aspirate of eight patients with primary plasma cell leukemia and isolated via Ficoll centrifugation. CD38 upregulation was done by incubating with 10 or 25 nM panobinostat for 24 h and the apoptosis detection employed Annexin V and Propidium Iodide staining followed by flow cytometry analysis. Results: A series of CLICK-able mAbs and semitelechelic polymer-drug conjugates were synthesized. Among them, we firstly prepared Epirubicin (EPI)-based pADCs, such as DARA-P-EPI, ISA-P-EPI and DRO-P-EPI (Fig.1A). Unlike naked mAbs, pADCs can induce EPI concentration-dependent cytotoxicity. Surface calreticulin (CRT) was assessed and demonstrated the induction of EPI-dependent immunogenic cell death on MM.1S cells (Fig.1B). The pADC mechanism of action involves binding to cell surface antigens, internalization via endosomes, and subsequent cleavage of drugs at lysosomes by cathepsin B. This minimizes premature drug release and reduces off-target toxicity (Fig.1C). Ex vivo studies on patient MM cells revealed that pADCs induced significantly higher apoptosis compared to naked mAbs and free EPI, highlighting their efficacy (Fig.1D). CD38-based pADC's efficacy was enhanced by pretreatment with panobinostat (Fig.1F), which increased CD38 expression (Fig.1E). When incubating with NK cells and pADC, the number of target cells decreased significantly indicating the pADC preserve the original mAb ADCC functionality. Notably, co-treatment of RPMI8226 cells with anti-CD38 and GDC0980, a PI3k/mTOR inhibitor, significantly improved cytotoxicity, suggesting DARA's potential for chemo sensitization (Fig.1G). Conclusion: pADC's innovative design offers a high likelihood of successful clinical development and holds significant promise for MM therapy. Moreover, the unique mechanism provides potential to effectively overcome mAb and multiple drug resistance. Its facile nature allows for easy incorporation of multiple types of payloads into the ADC, making its synthesis highly scalable .

Tris(2-carboxyethyl)phosphine-mediated immobilization of thiolated DNA on gold nanoclusters and its application in multiplex microRNA detection

Quick and efficient immobilization of DNAs on gold nanoclusters (AuNCs) has a substantial impact on nanoscale applications relative to molecular recognition and patterned assembly. We found that in the presence of tris(2-carboxyethyl)phosphine (TCEP), chemisorption of thiolated DNA on the AuNC surface could be accomplished within 1.5 h, which was dramatically shorter than the incubation time needed in the conventional thiolate exchange reactions. Based on this observation, a core-shell-satellite nanocomposite consisting of magnetic nanoparticle, silica coating, and AuNCs (Fe3O4NPs@SiO2@AuNCs) was fabricated, on which a fluorescent hairpin probe was anchored with the assistance of TCEP. Integrated with duplex-specific nuclease (DSN)-assisted target recycling strategy, the sensing platform could simultaneously detect microRNA-21 and microRNA-141 with detection limits of 0.020 and 0.017 pM, respectively. The feasibility of the developed immobilization approach might significantly advance the use of DNAs and AuNCs in analytical sciences and clinical research.

Disulfide Click Reaction for Stapling of S-terminal Peptides.

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC50 =6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.

Disulfide Click Reaction for Stapling of S‐terminal Peptides

AbstractA disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17‐membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S‐terminal peptides from 3 to 18 amino acid residues to provide 18‐ to 48‐membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti‐HCT‐116 activity with a locked α‐helical conformation (IC50=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2‐carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three‐dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.

Chemical-chemical redox cycling for improving the sensitivity of the fluorescent assay: A proof-of-concept towards DNA methylation detection

Ultrasensitive analytical methods are still urgent for the discovery of trace level biomarkers and the early clinical diagnosis of disease. In this work, an ultrasensitive universal sensing platform was constructed by integrating fluorescent assay with chemical-chemical redox cycling signal amplification strategy. Using Ru@SiO2 nanoparticles wrapped by MnO2 nanosheets (Ru@SiO2@MnO2) as fluorescent probe, the chemical-chemical redox cycling system was conducted upon ascorbic acid (AA) and tris(2-carboxyethyl)phosphine (TCEP) as reductants and MnO2 nanosheets as oxidant. The MnO2 nanosheets not only could quench the fluorescence of Ru@SiO2 nanoparticles to reduce the background, but also could serve as oxidants to react with AA, generating dehydroascorbic acid (DHA). The DHA was reduced by TCEP in turn to form AA that participated in the next cycling of chemical-chemical redox reaction. Thus, the constantly released AA from the chemical-chemical redox cycling system could massively etch MnO2 nanosheets on Ru@SiO2 surface, making the fluorescence of Ru@SiO2 nanoparticles greatly recovered. It was shown that the sensitivity of the fluorescent assay was improved almost 52 times by utilizing the chemical-chemical redox cycling signal amplification strategy. This strategy was further employed to detect DNA methylation with the aid of AA-encapsulated liposomes that were modified with 5 mC antibodies to bind with the methylated DNA captured in 96-well plate. A detection of limit down to 16.2 fM was achieved for the detection of methylated DNA. It's believed that the incorporation of chemical-chemical redox cycling signal amplification strategy into fluorescent sensing paves a new way for ultrasensitive detection of biomarkers.

Late-stage diversification strategy for the synthesis of peptide acids and amides using hydrazides

Aim: Modification of the C-terminus of a peptide to improve its properties, particularly after constructing the peptide chain, has great promise in the development of peptide therapeutics. This study discusses the development of a late-stage diversification method for synthesizing peptide acids and amides from hydrazides which can serve as a common precursor. Methods: Peptide hydrazides were synthesized solely by using conventional solid-phase peptide synthesis (SPPS). Hydrazides were subjected to oxidation by potassium peroxymonosulfate (Oxone) to afford carboxylic acids. Azidation of hydrazides using sodium nitrite (NaNO2) under acidic conditions, followed by the addition of β-mercaptoethanol (BME), could also be used to generate carboxylic acids. For the preparation of peptide amides, azides that can be prepared from hydrazides were reacted with ammonium acetate (NH4OAc) or tris(2-carboxyethyl)phosphine (TCEP)∙hydrochloride (HCl) to develop the products through ammonolysis or a Staudinger reaction, which produces iminophosphorane from an azide and a phosphine. The antimicrobial activity of modelin-5 derivatives synthesized from the corresponding hydrazides was evaluated by the colony count of Escherichia coli (E. coli) after treatment with the peptides. Results: Oxone oxidation yielded the corresponding acids rapidly although oxidation-prone amino acids were incompatible. Azidation and subsequent treatment with BME afforded peptide acids an acceptable yield even in sequences containing amino acids that are prone to oxidation. Both methods for conversion of hydrazides to amides were found to afford the desired products in good yield and compatibility. The conditions that were developed were adapted to the synthesis of modelin-5 derivatives from the corresponding hydrazides, yielding late-stage production of the desired peptides. The amides of the resulting peptide showed more potent activity against E. coli than the acid form, and the most potent activity was observed from the hydrazide. Conclusions: The developed protocols allow hydrazides to be converted to acids or amides, enabling late-stage diversification of peptide C-terminal residues.

A method for the quantitative and reversible trapping of sulfidic gases from headspaces and its application to the study of wine reductive off-odors.

Some relevant food systems release tiny amounts of sulfidic gases, whose measurement is difficult because of their inherent instability. The present paper demonstrates that Cu(I) solutions trap quantitatively and stabilize sulfidic gases. Once trapped, the gases remain stable for weeks at 4°C and at least 8days at 75°C. Trapped gases can be quantitatively released with tris(2-carboxyethyl) phosphine (TCEP) and brine dilution and then determined by GC. Trapping solutions, placed in 20-mL opened vials housed in 100mL hermetically-sealed flasks containing wine in anoxia, have been used to monitor the release of sulfidic gases by wines, revealing that at 50°C, up to 400μg/L of H2S and 58μg/L of MeSH can be released in 68days, and 3-5 times more at 75°C in 28days. The possibility to differentiate between released and accumulated amounts provides key clues to understanding the fate of sulfidic gases in wine and other food systems.

One-pot sample preparation procedure for the determination of protein N-linked homocysteine by HPLC-FLD based method

The report presents robust and high throughput method, based on liquid chromatography coupled with fluorescence detection (HPLC-FLD), for the determination of total protein N-linked homocysteine (Hcy) in human plasma. The assay involves simultaneous proteins precipitation with perchloric acid and removal of any other form of Hcy, except protein N-linked Hcy, via disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and plasma protein pellet wash with perchloric acid followed by liberation of N-linked Hcy from proteins by hydrochloric acid hydrolysis, drying under vacuum and residue reconstitution in diluted hydrochloric acid. The chromatographic separation of resulting in this way Hcy-thiolactone (HTL) is achieved within 3 min at room temperature on PolymerX RP-1 (150 × 4.6 mm, 5.0 µm) column using isocratic elution with eluent, consisted of o-phthaldialdehyde (OPA) in sodium hydroxide and acetonitrile (ACN), delivered at a flow rate 1 mL/min. The analyte is quantified by monitoring fluorescence at 480 nm using excitation at 370 nm, in a linear range from 0.25 to 10 µmol/L in plasma, while the limit of quantification (LOQ) equals 0.25 µmol/L. The method was successfully applied to plasma samples delivered by fifteen apparently healthy donors showing that the HPLC-FLD assay is suitable for screening of human plasma.

Simultaneous determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid and main plasma aminothiols by HPLC–UV based method

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5′-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC–UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1–100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC–UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.