Thiol Peptides Research Articles

Overexpression of bacterial γ-glutamylcysteine synthetase increases toxic metal(loid)s tolerance and accumulation in Crambe abyssinica.

Transgenic Crambe abyssinica lines overexpressing γ-ECS significantly enhance tolerance to and accumulation of toxic metal(loid)s, improving phytoremediation potential and offering an effective solution for contaminated soil management. Phytoremediation is an attractive environmental-friendly technology to remove metal(loid)s from contaminated soils and water. However, tolerance to toxic metals in plants is a critical limiting factor. Transgenic Crambe abyssinica lines were developed that overexpress the bacterial γ-glutamylcysteine synthetase (γ-ECS) gene to increase the levels of non-protein thiol peptides such as γ-glutamylcysteine (γ-EC), glutathione (GSH), and phytochelatins (PCs) that mediate metal(loid)s detoxification. The present study investigated the effect of γ-ECS overexpression on the tolerance to and accumulation of toxic As, Cd, Pb, Hg, and Cr supplied individually or as a mixture of metals. Compared to wild-type plants, γ-ECS transgenics (γ-ECS1-8 and γ-ECS16-5) exhibited a significantly higher capacity to tolerate and accumulate these elements in aboveground tissues, i.e., 76-154% As, 200-254% Cd, 37-48% Hg, 26-69% Pb, and 39-46% Cr, when supplied individually. This is attributable to enhanced production of GSH (82-159% and 75-87%) and PC2 (27-33% and 37-65%) as compared to WT plants under AsV and Cd exposure, respectively. The levels of Cys and γ-EC were also increased by 56-67% and 450-794% in the overexpression lines compared to WT plants under non-stress conditions, respectively. This likely enhanced the metabolic pathway associated with GSH biosynthesis, leading to the ultimate synthesis of PCs, which detoxify toxic metal(loid)s through chelation. These findings demonstrate that γ-ECS overexpressing Crambe lines can be used for the enhanced phytoremediation of toxic metals and metalloids from contaminated soils.

Organic Synthesis Away from Equilibrium: Contrathermodynamic Transformations Enabled by Excited-State Electron Transfer.

ConspectusChemists have long been inspired by biological photosynthesis, wherein a series of excited-state electron transfer (ET) events facilitate the conversion of low energy starting materials such as H2O and CO2 into higher energy products in the form of carbohydrates and O2. While this model for utilizing light-driven charge transfer to drive catalytic reactions thermodynamically "uphill" has been extensively adapted for small molecule activation, molecular machines, photoswitches, and solar fuel chemistry, its application in organic synthesis has been less systematically developed. However, the potential benefits of these approaches are significant, both in enabling transformations that cannot be readily achieved using conventional thermal chemistry and in accessing distinct selectivity regimes that are uniquely enabled by excited-state mechanisms. In this Account, we present work from our group that highlights the ability of visible light photoredox catalysis to drive useful organic transformations away from their equilibrium positions, addressing a number of long-standing synthetic challenges.We first discuss how excited-state ET enabled the first general methods for the catalytic anti-Markovnikov hydroamination of unactivated alkenes with alkyl amines. In these reactions, an excited-state iridium(III) photocatalyst reversibly oxidizes secondary amine substrates to their corresponding aminium radical cations (ARCs). These electrophilic N-centered radicals can then react with olefins to furnish valuable tertiary amine products with complete anti-Markovnikov regioselectivity. Notably, some of these products are less thermodynamically stable than their corresponding amine and alkene starting materials. We next present a strategy for light-driven C-C bond cleavage within various aliphatic alcohols mediated by homolytic activation of alcohol O-H bonds by excited-state proton-coupled electron transfer (PCET). The resulting alkoxy radical intermediates then undergo C-C β-scission to ultimately provide isomeric linear carbonyl products that are often higher in energy than their cyclic alcohol precursors. Applications of this chemistry for the light-driven depolymerization of lignin biomass, commercial phenoxy resin, hydroxylated polyolefin derivatives, and thermoset polymers are presented as well. We then describe a method for the contrathermodynamic positional isomerization of highly substituted olefins by means of cooperative photoredox and chromium(II) catalysis. In this work, generation of an allylchromium(III) species that can undergo highly regioselective in situ protodemetalation enables access to a less substituted and thermodynamically less stable positional isomer. Product selectivity in this reaction is determined by the large differential in oxidation potentials between differently substituted olefin isomers. Lastly, we discuss a light-driven deracemization reaction developed in collaboration with the Miller group, wherein a racemic urea substrate undergoes spontaneous optical enrichment upon visible light irradiation in the presence of an iridium(III) chromophore, a chiral Brønsted base, and a chiral peptide thiol. Excellent levels of enantioselectivity are achieved via sequential and synergistic proton transfer (PT) and H atom transfer (HAT) steps. Taken together, these examples highlight the ability of excited-state ET events to enable access to nonequilibrium product distributions across a wide range of catalytic, redox-neutral transformations in which photons are the only stoichiometric reagents.

Cryo-EM structure of cadmium-bound human ABCB6

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity.

The effect of sulphur supplementation on cadmium phytotoxicity in wheat and lettuce: changes in physiochemical properties of roots and accumulation of phytochelatins.

Intensive sulphur fertilisation has been reported to improve the nutrient balance and growth of Cd-exposed plants, but the reasons of this phenomenon and the role of sulphur compounds in the resistance to cadmium are unclear. We investigated sulphur supplementation-induced changes in the surface properties of roots and the level of thiol peptides (PCs) in Cd-stressed Triticum aestivum L. (monocots clade) and Lactuca sativa L. (dicots clade) grown in nutrient solution. The combination of three sulphur (2mM S-basic level, 6 or 9mM S-elevated levels) and four cadmium (0, 0.0002, 0.02 or 0.04mM Cd) concentrations was used. The physicochemical parameters of the roots were determined based on the apparent surface area (Sr), total variable surface charge (Q), cation exchange capacity (CEC) and surface charge density (SCD). In Cd-exposed plants supplied with sulphur, a different character and trend in the physicochemical changes (adsorption and ion exchange) of roots were noted. At the increased sulphur levels, as a rule, the Sr, CEC, Q and SCD values clearly increased in the lettuce but decreased in the wheat in the entire range of the Cd concentrations, except the enhanced Sr of wheat supplied with 6mM S together with elevated (0.0002mM) and unchanged (0.02, 0.04mM Cd) value of this parameter at 9mM S. This indicates a clade-specific and/or species-specific plant reaction. The 6mM S appears to be more effective than 9mM S in alleviation of the cadmium's toxic effects on roots. It was found that at 0.02 and 0.04mM Cd, the use of 6mM S limits the Cd accumulation in the roots of both species in comparison with the basic S fertilisation. Moreover, PC accumulation was much more efficient in wheat than in lettuce, and intensive sulphur nutrition generally induced biosynthesis of these chelating compounds. Physicochemical parameters together with quantitative and qualitative assessment of thiol peptides can be important indicators of the efficiency of root system functioning under cadmium stress. The differences between the species and the multidirectional character of the changes are a result of the involvement of a number of multi-level mechanisms engaged in the defence against metal toxicity.

Cystine/Glutamate antiporter system xc- deficiency impairs macrophage glutathione metabolism and cytokine production.

System xc-, encoded by Slc7a11, is an antiporter responsible for exporting glutamate while importing cystine, which is essential for protein synthesis and the formation of thiol peptides, such as glutathione. Glutathione acts as a co-factor for enzymes responsible for scavenging reactive oxygen species. Upon exposure to bacterial products, macrophages exhibit a rapid upregulation of system xc-. This study investigates the impact of Slc7a11 deficiency on the functionality of peritoneal and bone marrow-derived macrophages. Our findings reveal that the absence of Slc7a11 results in significantly reduced glutathione levels, compromised mitochondrial flexibility, and hindered cytokine production in bone marrow-derived macrophages. Conversely, system xc- has a lesser impact on peritoneal macrophages in vivo. These results indicate that system xc- is essential for maintaining glutathione levels, mitochondrial functionality, and cytokine production, with a heightened importance under atmospheric oxygen tension.

Glutathione and phytochelatins jointly allow intracellular and extracellular detoxification of cadmium in the liverwort Marchantia polymorpha

Plants have evolved a set of mechanisms that control and respond to the uptake and accumulation of both essential and non-essential metals, including chelation and sequestration of these elements by thiol ligands, such as glutathione and phytochelatins. Indeed, such thiol peptides can chelate some metals, quickly form thiol-metal complexes, and segregate them in the vacuolar compartment. Reasonably, conceptually similar mechanisms can be assumed to be responsible for the transport of metal complexes ̶ in particular thiol-cadmium complexes ̶ across the plasma membrane, with the consequent release of this toxic metal in the extracellular environment. Such hypothesis, focusing on prevention and detoxification mechanisms, was here verified in axenically-grown gametophytes of the model liverwort Marchantia polymorpha, exposed to three different cadmium concentrations over five exposure times. From the data obtained, it can be deduced that the cell wall of M. polymorpha moderately reduced the influx of cadmium into the cells, since it was rapidly saturated by this metal. At an intracellular level, cadmium induced the activity, but not the gene expression, of the phytochelatin synthase enzyme, leading to synthesis of phytochelatins. Moreover, both glutathione and phytochelatins chelated cadmium at the cytosolic level and allowed its detoxification, possibly involving tonoplast transporters belonging to type-C ABC subfamily (Mp7g13860 and Mp4g11930). Likewise, cadmium, glutathione and phytochelatins were released extracellularly, thus highlighting a possible novel role in cadmium detoxification, in a pH-dependent manner. The overall results suggest that, in M. polymorpha, glutathione and phytochelatins can accomplish intracellular and extracellular detoxification of cadmium.

Peptide Cysteine Thiols Act as Photostabilizer of Avobenzone through Stabilizing the Transition State of Keto-Enol Tautomerization.

Photostabilizers have been used to impart stability to an FDA-approved chemical UV-A filter avobenzone against the UV-A radiations and sunlight. The thiol group of glutathione plays a critical role in imparting the photostabilization activity of glutathione on avobenzone. The current report aims to evaluate the photostabilization activity of multiple thiols containing cysteine peptides on avobenzone. Cysteine-tripeptide and cysteine-pentapeptide were chemically synthesized and characterized using mass spectrometry. Synthetic peptides were assessed for their photostabilization activity on the enolic-form of the avobenzone under natural sunlight using UV spectroscopy in both protic and aprotic solvents. Unlike glutathione, which has pronounced activity in protic solvents, cysteine-pentapeptide exhibits similar photoprotection activity in both protic and aprotic solvents. Computational calculations using DFT suggest that peptide cysteine thiols may assist in the reversal of the photoketonization process of avobenzone thereby exhibiting the photoprotection activity to the enolic-form of avobenzone. Peptide cysteine thiols lower the activation energy barrier of keto-to-enol tautomerization of avobenzone by 30 kcal mol-1 by assisting the proton shuttle through a six-membered transition state. The current report emphasizes the applications of peptide thiols in cosmetics and may help in the development of peptides as aesthetic medicines.

Multifunctional DNA mediated spatially confined assembly for antibody orientation: Surpassing sensitivity and accuracy for rituximab detection

The sensitive, accurate and fast monitoring of therapeutic drugs plays an important role in guiding clinical medication or individualized treatment. Inspired by the highly efficient interactions of ordered structures in living organisms, a multifunctional DNA mediated spatially confined assembly (SCA) based antibody orientation strategy was proposed in this work. The multifunctional DNA containing a signaling molecule labeled on the head and a poly adenine tail (20 bases), can not only direct the ordered immobilization of antibodies on gold nanoparticles, but can also enrich electrochemical signal molecules. Compared with a randomly arranged antibody immobilization strategy, the SCA based antibody orientation strategy can produce nearly 200% of peak current signal. Moreover, MoS2/gold nanoparticles were used to modify electrodes in this study in order to improve the electrochemically active surface area and electron transfer of the electrode as well as the spatial regulation of thiol peptides. By using a highly specific CD20 epitope mimetic peptide this proposed biosensor exhibits high sensitivity for rituximab detection (LOD 56.4 fM) as well as good selectivity, stability and satisfactory accuracy. When applying it for detecting rituximab in lymphoma patients’ plasma, comparable results to commercial ELISA were obtained with a low RSD (＜6.5%). The proposed signal tag fabrication strategy could easily be extended to analyze other targets by changing the sensing platform, which has a potential application in the fields of biological analysis and medical diagnosis.

Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear.The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial.We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.

Rhodium-Catalyzed Oxidation of Unprotected Peptide Thiols to Disulfides with Oxygen in Water

Unprotected peptide thiols are efficiently oxidized to peptide disulfides in homogeneous water. The oxidation reaction proceeds under ambient pressure of oxygen (1 atm), using a readily available R...

Ultrasensitive detection of CYFRA 21-1 DNA via SI-RAFT based in-situ metallization signal amplification

Cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA is a significant biomarker with relation to non-small cell lung cancer. Here, we described an ultrasensitive electrochemical biosensor for detection of CYFRA 21-1 DNA (target DNA, tDNA) via surface-initiated reversible addition fragmentation chain transfer (SI-RAFT) polymerization-based DNA metallization signal amplification strategy. Specifically, 5′terminal modified thiol peptide nucleic acid (PNA) probes are immobilized on the gold electrode surface (by Au-S selfassembly) for the identification of specific tDNA sequences. After hybridization, the 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPAD) is tethered to the PNA/DNA duplexes by means of the recognized phosphate-Zr4+-carboxylate chemistry. Subsequently, in the situation of C3H4O as the monomer, SI-RAFT polymerization can bring numerous aldehyde groups sites for the subsequent silver mirror reaction. The acrolein-polymers act as reductant to reduce Ag+ into Ag0, and then the silver particles are deposited on the polymer skeleton, which significantly amplifies the electrochemical signal. Under optimal conditions, the designed sensor for tDNA detection show a wide linear range from 100 aM to 1 nM, and the detection limit is as low as 0.89 aM. Moreover, the proposed biosensor exhibits prominent applicability for analysis of tDNA in serum samples and high selectivity for different mismatched oligonucleotide fragments. Given the proposed biosensor without extra natural enzyme or bio-labels, this method has enormous potential in bioanalysis.

The Moss Leptodictyum riparium Counteracts Severe Cadmium Stress by Activation of Glutathione Transferase and Phytochelatin Synthase, but Slightly by Phytochelatins.

In the present work, we investigated the response to Cd in Leptodictyum riparium, a cosmopolitan moss (Bryophyta) that can accumulate higher amounts of metals than other plants, even angiosperms, with absence or slight apparent damage. High-performance liquid chromatography followed by electrospray ionization tandem mass spectrometry of extracts from L. riparium gametophytes, exposed to 0, 36 and 360 µM Cd for 7 days, revealed the presence of γ-glutamylcysteine (γ-EC), reduced glutathione (GSH), and traces of phytochelatins. The increase in Cd concentrations progressively augmented reactive oxygen species levels, with activation of both antioxidant (catalase and superoxide dismutase) and detoxifying (glutathione-S-transferase) enzymes. After Cd treatment, cytosolic and vacuolar localization of thiol peptides was performed by means of the fluorescent dye monochlorobimane and subsequent observation with confocal laser scanning microscopy. The cytosolic fluorescence observed with the highest Cd concentrations was also consistent with the formation of γ-EC-bimane in the cytosol, possibly catalyzed by the peptidase activity of the L. riparium phytochelatin synthase. On the whole, activation of phytochelatin synthase and glutathione-S-transferase, but minimally phytochelatin synthesis, play a role to counteract Cd toxicity in L. riparium, in this manner minimizing the cellular damage caused by the metal. This study strengthens previous investigations on the L. riparium ability to efficiently hinder metal pollution, hinting at a potential use for biomonitoring and phytoremediation purposes.

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

The Oxidative Pathway to Dopamine-Protein Conjugates and Their Pro-Oxidant Activities: Implications for the Neurodegeneration of Parkinson's Disease.

Neuromelanin (NM) is a dark brown pigment found in dopaminergic neurons of the substantia nigra (SN) and in norepinephrinergic neurons of the locus coeruleus (LC). Although NM is thought to be involved in the etiology of Parkinson’s disease (PD) because its content decreases in neurodegenerative diseases such as PD, details are still unknown. In this study, we characterized the biosynthetic pathway of the oxidation of dopamine (DA) by tyrosinase in the presence of thiol peptides and proteins using spectroscopic and high-performance liquid chromatography (HPLC) methods and we assessed the binding of DA via cysteine residues in proteins by oxidation catalyzed by redox-active metal ions. To examine whether the protein-bound DA conjugates exhibit pro-oxidant activities, we measured the depletion of glutathione (GSH) with the concomitant production of hydrogen peroxide. The results suggest that the fate of protein-bound DA conjugates depends on the structural features of the proteins and that DA-protein conjugates produced in the brain possess pro-oxidant activities, which may cause neurodegeneration due to the generation of reactive oxygen species (ROS) and the depletion of antioxidants.

The use of micro-energy dispersive X-ray fluorescence spectrometry combined with a multivariate approach to determine element variation and distribution in tobacco seedlings exposed to arsenate

Here, we present a new scheme of analysis combining micro-energy dispersive X-ray fluorescence spectrometry (μ-XRF) with a multivariate approach that allows to establish the inter-correlation of multiple elements and their elemental map in plants. The main advantage of this procedure is that XRF spectral profiles can be analysed directly, by means of principal component analysis (PCA), allowing a quick interpretation of the results. Furthermore, this analysis requires small amounts of plant material and can be performed in whole individual seedlings in the hydrated state without chemical extraction. With this technology, we determined the distribution of arsenic (As) and the variation and spatial distribution of multiple elements in whole tobacco seedlings grown in the presence of different arsenate concentrations. We observed that As is detectable mainly in roots, primarily in the basal part, but also in the root apex in seedlings grown in the highest arsenate concentration. The low rate of As translocation from roots to shoots and the significant increase in S are consistent with previous evidence showing that As is retained in roots by forming complexes with thiol peptides. We also found a significant non-linear increase in P, as arsenate is taken up by phosphate transporters and induces the expression of genes encoding them. A decrease in Mn, Fe and Zn proportional to the accumulation of As and in the same tissues, suggests a competition of these elements with As for cellular transporters.

Comprehensive Redox Profiling of the Thiol Proteome of Clostridium difficile

The strictly anaerobic bacterium C. difficile has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most C. difficile infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of C. difficile, stress adaptation mechanisms and regulation of virulence factors is mandatory. The focus of this work was to tackle the thiol proteome of C. difficile and its stress-induced alterations, because recent research has reported that the amino acid cysteine plays a central role in the metabolism of this pathogen. We have developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of this technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of C. difficile We present redox-quantification for more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful not only when determining redox status, but also for providing information on protein quantity. Additionally, our comprehensive data set reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in C. difficile.

Fluorescent hypoxia probe for flow cytometry

Abstract Hypoxia is a condition where low levels of oxygen levels are present (1% to 2% O2). Hypoxia play a wide role in physiological and pathological conditions, from developmental angiogenesis to tumor progression and evasion. Hypoxia also modulates a number of immune functions from promoting inflammation to suppressing adaptive immunity. The current method for detecting hypoxic cells relies on an immunochemical approach. Pimonidazole react with peptide thiols in hypoxic cells and the resulting adduct is detected with an anti-pimonidazole antibody. To increase the availability of hypoxia detection reagents, we have developed a rhodamine-based hypoxia reagent (HR) for live cells. Using Jurkat leukemia cells incubated in hypoxic or normoxic conditions and stained with HR, we were able to clearly distinguish hypoxic cells from normoxic cells on a flow cytometer. We were also able to resolve cells incubated at differing levels of O2 (10%, 5% and 2.5% O2). To demonstrate that HR is compatible with other reagents, hypoxic cells were co-stained with a viability dye (SYTOX Red), a dye retained in intact mitochondria (TMRM) and with staining for a marker of apoptosis (annexin V). The results show that hypoxic cells excluded the dead cells dye while retaining TMRM, and were negative for annexin V staining at early time points. As expected, prolong incubation at hypoxia resulted in dead and apoptotic cells as evident in an increase in dead cells staining, a loss of mitochondrial integrity and an increase in annexin V+ cells. In summary, we present a sensitive reagent for detecting hypoxic cells without the need for cellular fixation and permeablization, while retaining it usage with other live cell flow cytometry detection reagents.

Glutathione is a highly efficient thermostabilizer of poliovirus Sabin strains

Glutathione (GSH) is the most abundant thiol peptide in animal cells and has a critical role in antioxidation. GSH was reported to be essential for stabilization of some enteroviruses, including poliovirus (PV), during viral morphogenesis. Here, we explored the potential use of GSH as a thermostabilizer of oral poliomyelitis vaccine (OPV) formulations. GSH significantly protected the three types of PV from heat-inactivation in a concentration-dependent manner. At a GSH concentration of 20mM, nearly complete protection was observed against heating temperatures up to 53°C for 2min.GSH also markedly protected PV1 from heat-inactivation and this up to 6 h at temperatures of 44°C and 46°C and 3 h at 48°C. The fact that GSH is naturally present at high concentration in the human body makes it an efficient candidate stabilizer for OPV formulations.

Electrochemical assay of proteolytically active prostate specific antigen based on anodic stripping voltammetry of silver enhanced gold nanoparticle labels

This work demonstrated the determination of proteolytically active prostate specific antigen (paPSA), a potential biomarker for prostate cancer diagnosis, in human serum using a sensitive and low-cost electrochemical sensor. A specifically designed peptide probe was immobilized on the surface of a 96-well plate. The probe could be recognized by paPSA causing cleavage of the peptide, resulting in a decrease in the thiols group remaining on the probe. Gold nanoparticles (AuNPs) were attached to the peptide thiol groups by self-assembly. Hence the amount of AuNPs relates to the length of peptide probe. After cleavage and binding of AuNPs, an amplification step was performed using a silver enhancer solution. The quantity of deposited silver was then measured by differential pulse anodic stripping voltammetry (DPASV) using a disposable screen-printed carbon electrode (SPCE). The signal for paPSA detection was the linear range from 0.1 to 100ngmL−1, with a detection limit of 27pgmL−1. We also showed that the assay was reliable and has potential for clinical applications.

Physiological mechanisms of adaptation of Dianthus carthusianorum L. to growth on a Zn-Pb waste deposit - the case of chronic multi-metal and acute Zn stress

This study investigates the response of metallicolous (M) and nonmetallicolous (NM) ecotypes of Dianthus carthusianorum L. to chronic multi-metal and acute Zn stress. Plants were cultivated on the Zn-Pb waste heap substrate and under Zn excess in hydroponics. Growth parameters as well as accumulation of organic acids and thiol peptides were determined as a function of metal accumulation. When grown on the metalliferous substrate, the M plants showed less phytotoxicity symptoms, lower foliar metal (Zn, Pb, Cd) accumulation, higher malate and citrate but lower glutathione content than the NM plants. When exposed to Zn excess in hydroponics, the M ecotype was also more tolerant but accumulated more Zn in comparison with the NM ecotype, accompanied by greater malate and citrate concentrations in the shoots, which were however not affected by increasing Zn doses. No phytochelatins were detected under any experimental conditions. Both constitutive and adaptive tolerance was found in D. carthusianorum. Under chronic metal stress, enhanced tolerance results from restricted metal uptake to the shoots and probably from detoxification by organic acids; however, under acute Zn stress it is not related to diminished metal uptake or organic acids. Glutathione and phytochelatins are not implicated in adaptive metal tolerance.