Stem cell‐based therapy has been highlighted as a potential avenue to promote tissue regeneration, where stimulation of stem cells to differentiate into the targeted cell type is essential. One of ...the factors that induce stem cells to differentiate is their surrounding microenvironment. In this study, the correlation between mild reductant and early osteogenic commitment was evaluated. A cell surface‐reducing microenvironment significantly silenced the transforming growth factor (TGF)‐β signaling pathway of mesenchymal stem cells (MSCs), followed by increased focal adhesion and inhibition of cell membrane protein dimerization. Furthermore, in vivo transplantation of MSCs exposed to the reducing microenvironment resulted in an early osteogenic commitment and neobone formation. Thus, these results highlight the potential of cell surface‐reducing microenvironment to influence early osteogenic commitment.
Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due ...to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.
The objective of the study was to develop a new method for the determination of the total content of vitamin C and dehydroascorbic acid in food, based on the technique of differential pulse ...voltammetry with the use of a boron-doped diamond electrode modified with mercury film. A comparison was made between the results obtained with the developed method and a proposed reference method based on high-performance liquid chromatography with spectrophotometric detection. The reduction of dehydroascorbic acid was performed with the use of tris(2-carboxyethyl)phosphine. The interference caused by the presence of tris(2-carboxyethyl)phosphine during the voltammetric determination of ascorbic acid was effectively eliminated through a reaction with N-ethylmaleimide. The conducted validation of the voltammetric method indicated that correct results of analysis of the total content of vitamin C and ascorbic acid were obtained. Analysis of the content of dehydroascorbic acid was imprecise due to the application of the differential method. The results of the analyses and the determined validation parameters of the developed method are characterised by a high degree of conformance with the results obtained with the chromatographic reference method, which indicates the equivalence of the two methods.
Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. ...Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.
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•Polyclonal half-antibody production has been investigated.•Reduction mixtures containing half-antibody fragments might contain other products.•Higher half-antibody fragment yields were obtained by manipulating antibody host, pH, reductant and its concentration.•During the course of the reduction reaction characteristic mixtures of antibody fragments were obtained.•Biologically active half-antibody fragment production from rabbit polyclonal anti-myoglobin IgG has been optimized.
Recent studies reveal a great value of interleukin-8 (IL-8), a pro-inflammatory cytokine, as a potent biomarker for early diagnosis of oral cancer. Herein, a new electrochemical method is proposed to ...detect IL-8 by facilely incorporating DNA-templated quantum dots (QDs). In principle, target IL-8 is first treated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to yield active thiols and then captured by antibody-functionalized magnetic beads (MBs). Thereafter, via the Michael addition reaction between the active thiol and maleimide group, a maleimide-modified DNA probe is linked to the surface of MBs, which can initiate a process of rolling circle amplification. In this way, long-range DNA strands are generated on the MB surface, subsequently recruiting DNA-templated CdTe/CdS QDs (DNA-QDs) to act as electrochemical reporters. By tracing the responses of DNA-QDs, the method allows IL-8 detection in a linear range from 5 to 5000 fg/mL with a detection limit of 3.36 fg/mL. The selectivity, reproducibility, and applicability in complex serum samples are also demonstrated to be favorable, indicating that the method may have a great potential in the future. More importantly, the use of TCEP treatment in the method not only provides a facile way to incorporate DNA-QDs, avoiding the complicated and time-consuming preparation process of antibody-DNA conjugates or functional nanomaterials; but also makes the method capable of being extended to detect other protein biomarkers in view of widespread presence of disulfides, which may hold a broad potential to facilitate efficient biosensing designs.
Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline ...hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.
•Freeze-drying enhances extraction of anthocyanins and vitamin C from potato tubers.•Steam-cooking stabilizes ascorbic acid in potato tubers by enzyme deactivation.•In raw potatoes total vitamin C ...can be reliably quantified but not ascorbic acid.•Tris(2-carboxyethyl) phosphine hydrochloride reduces dehydroascorbic acid efficiently.
Methods were optimized for extraction and quantification of anthocyanins (ACY) and vitamin C in potatoes. Acidified aqueous methanol (70%) was the optimal extraction solvent and freeze-drying significantly improved the extraction yield of ACY. The content of ACY varied widely in five potato cultivars from 0.42 to 3.18mg/g dry weight, with the latter being the highest value found in the Finnish cultivar ‘Synkeä Sakari’.
Compared with dithiothreitol (DTT), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was more efficient in reducing dehydroascorbic acid (DHA) to ascorbic acid (AA) and for quantifying the content of total ascorbic acid (TAA). For raw potatoes, quantification of TAA after treatment with TCEP was more reliable than a direct analysis of AA, whereas AA can be analyzed directly in steam-cooked samples. The TAA contents in the three potato cultivars were around 0.30–0.35mg/g dry weight. The loss of AA in steam cooking was 24%.
The analysis of total vitamin C content in food is most frequently performed by reducing dehydroascorbic acid to ascorbic acid, which is then assayed with the technique of high-performance liquid ...chromatography combined with spectrophotometric detection. Tris(2-carboxyethyl)phosphine is currently the only agent in use that efficiently reduces dehydroascorbic acid at pH < 2. Therefore, there is a continued need to search for new reducing agents that will display a high reactivity and stability in acidic solutions. The objective of the study was to verify the applicability of unithiol and tris(hydroxypropyl)phosphine for a reducing dehydroascorbic acid in an extraction medium with pH < 2. The conducted validation of the newly developed method of determining the total content of vitamin C using tris(hydroxypropyl)phosphine indicates its applicability for food analysis. The method allows obtaining equivalent results compared to the method based on the use of tris(2-carboxyethyl)phosphine. The low efficiency of dehydroascorbic acid reduction with the use of unithiol does not allow its application as a new reducing agent in vitamin C analysis.
The influence of the introduction of tris(2-carboxyethyl)phosphine on the dyeing of wool with natural dyes was studied and the kinetics and thermodynamics of the dyeing process were also ...investigated. None of the previous techniques employed report the possibility of modification of wool fibers with tris(2-carboxyethyl)phosphine for dyeing. The modified wool fabrics represented important advances in dye adsorption and decrease in dye desorption at a low dyeing temperature (80 °C) that are not available in the other systems without metallic mordants. Our results demonstrated that the adsorption of natural dyes on wool fabrics was greatly improved through the selective reduction of disulphide bonds with tris(2-carboxyethyl)phosphine. In addition, experiment data was analyzed using pseudo-second order kinetics and Langmuir type isotherms. The results indicated that the diffusion coefficient of dyes was increased while the dyeing entropy and enthalpy were decreased with the introduction of tris(2-carboxyethyl)phosphine.
•Tris(2-carboxyethyl)phosphine was introduced in the dyeing of wool fabrics.•The dyeing wool fabrics exhibited high dye adsorption and low dye desorption.•The diffusion coefficient of dyes was increased with the addition of tris(2-carboxyethyl)phosphine.•The dyeing entropy and enthalpy were decreased with the addition of tris(2-carboxyethyl)phosphine.
The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It ...is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)Cl(-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)Cl(-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score.
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