Search Results (107)

Polymer coatings as thin films stand out as a commonly used strategy to modify biosensor surfaces for improving detection performance; however, nonspecific biomolecule interactions and the limited degree of ligand conjugation on the surface have necessitated the development of innovative methods for surface modification. To this end, methacrylated tethered telechelic polyethylene glycol (PEG-diMA) chains of three different molecular weights (2, 6, and 10 kDa) were synthesized herein and used for obtaining thiolated nanoparticles (NPs) upon adding excess amounts of a tetra-thiol crosslinker. Characterized according to their size, surface charge, morphology, and thiol amounts, these nanoparticles were immobilized on gold surfaces that mimicked gold-coated mass sensor platforms. The PEG-based nanoparticles, prepared especially by PEG6K-diMA polymers, were shown to result in the preparation of a monolayer and smooth coating of 80–120 nm thickness. Cysteine-modified NTS(8–13) peptide (RRPYIL) was conjugated to thiolated NP with reversible disulfide bonds and it was demonstrated that its cleavage with a reducing agent such as dithiothreitol (DTT) restores the NP-immobilized gold surface for at least two cycles. Together with its binding studies to NTSR2 antibodies, it was revealed that the peptide-conjugated NP-modified gold surface could be employed as a model for a reusable sensor surface for the detection of biomarkers of same or different types. Full article

Background: The thioredoxin system (TrxS) is crucial for maintaining redox balance by regulating cellular thiol levels and is involved in various biological processes, including cancer progression. Thioredoxin reductase (TrxR), a key component of TrxS, reduces oxidized thioredoxin (Trx) using NADPH. This study investigates the inhibitory effects of 2-bromo-2-nitro-1,3-propanediol (Bronopol, BP), a preservative, on TrxR activity and its impact on cellular thiols and cell viability. Methods: Purified recombinant TrxR and noncancer and cancer cells were treated with different concentrations of BP and TrxR activity measured. BP-treated cells were examined for effects of BP on total cellular thiol level and GSH/GSSG ratio. Results: BP effectively inhibited TrxR in a dose-dependent manner, an effect that was reversible with dithiothreitol (DTT). BP treatment significantly reduced total thiol levels, decreased the GSH/GSSG ratio, and increased reactive oxygen species (ROS) in cells. Additionally, BP decreased cell viability and induced apoptosis, as indicated by morphological changes and increased c-fos mRNA expression. Conclusions: These findings highlight BP’s potential as a TrxR inhibitor and its cytotoxicity toward both noncancer and cancer cells. The observed effects—TrxR inhibition, thiol oxidation, GSH/GSSG imbalance, and ROS accumulation—may underlie BP’s cytotoxicity. Further research is needed to explore the precise molecular mechanisms by which BP exerts these effects. Full article

Chlorella, recognized for its high lipid and protein content, is increasingly studied for its potential in the food and bio industries. To enhance its production and understand the underlying mechanisms of lipid accumulation, this study investigated the role of endoplasmic reticulum (ER) stress in modulating lipid metabolism in Chlorella sorokiniana UTEX 2714, using six putative ER stress inducers: 2-deoxy-D-glucose (2-DG), dithiothreitol (DTT), tunicamycin (TM), thapsigargin (TG), brefeldin A (BFA), and monensin (Mon). The results showed that 2-DG, DTT, TM, BFA, and Mon significantly inhibited cell growth in C. sorokiniana. Treatment with 2-DG, DTT, TM, BFA, or Mon resulted in substantial increases in the triacylglycerol (TAG) to total fatty acid (tFA) ratio, with fold changes of 14.8, 7.9, 6.2, 10.1, and 8.9, respectively. Among the tFAs, cells treated with these compounds exhibited higher levels of saturated fatty acids and lower levels of polyunsaturated fatty acids (PUFAs). In contrast, the fatty acid composition of TAGs showed the opposite trend, with relative enrichment in PUFAs. This study enhances our understanding of Chlorella lipid metabolism, providing valuable insights for optimizing lipid production, particularly TAGs enriched with PUFA content, for applications in functional foods, nutraceuticals, and sustainable bioresources. Full article

This study examines long-term trends in fine particulate matter (PM_2.5) composition and oxidative potential in Los Angeles based on data from the University of Southern California’s Particle Instrumentation Unit, with chemical composition retrieved from the EPA’s Air Quality System (AQS). While regulatory interventions have reduced PM_2.5 mass concentration and primary combustion-related components, our findings reveal a more complex toxicity pattern. From 2001 to 2008, the PM_2.5 oxidative potential, measured via the dithiothreitol (DTT) assay, declined from ~0.84 to ~0.16 nmol/min/m³ under stringent tailpipe controls. However, after this initial decline, PM_2.5 DTT stabilized and gradually increased from ~0.35 in 2012 to ~0.97 nmol/min/m³ by 2024, reflecting the growing influence of non-tailpipe emissions such as brake/tire wear. Metals, such as iron (Fe, ~150 ng/m³) and zinc (Zn, ~10 ng/m³), remained relatively stable as organic and elemental carbon (OC and EC) declined, resulting in non-tailpipe contributions dominating PM_2.5 toxicity. Although PM_2.5 mass concentrations were effectively reduced, the growing contribution of non-tailpipe emissions (e.g., brake/tire wear and secondary organic aerosols) underscores the limitations of mass-based standards and tailpipe-focused strategies. Our findings emphasize the need to broaden regulatory strategies, targeting emerging sources that shape PM_2.5 composition and toxicity and ensuring more improvements in public health outcomes. Full article

Environmental particulate matter (PM) exposure has been widely recognized for its significant adverse effects on human health. Monitoring PM levels is one of the essential parameters of air quality assessment. However, PM mass concentration alone does not sufficiently explain its toxicological impacts and effects on health. This study highlights the importance of oxidative potential (OP) as a promising metric for evaluating PM toxicity. It focuses on standardizing the dithiothreitol (DTT) assay as a tool for OP measurement. In order to investigate the impact of various extraction techniques, reagent concentrations, and assay conditions, four previously established protocols were tested without modification, while a novel protocol was introduced based on an extensive literature review. Results revealed strong positive correlations between the new and most established protocols. These findings highlight the significance of the new protocol in advancing the development of standardized methodologies for applying the DTT assay and demonstrating its reliability and relevance. While developing a standardized DTT assay involves addressing numerous parameters—from filter extraction to assay application—this research provides a solid base for achieving consistency in OP measurements and overcoming this critical issue. Full article

With the rationale that the oxidative potential of particulate matter (PM-OP) may induce oxidative stress and inflammation, we conducted the ASTHMA-FENOP study in which 44 asthmatic patients and 37 matched controls wore a personal sampler for 24 h, allowing the collection of fine and coarse PM fractions separately, to determine PM-OP by the dithiothreitol (DTT) and ascorbic acid (AA) methods. The levels of Interleukin 6 (IL-6) and the IL-6/IL-10 ratio, as indicators of pro- and anti-inflammatory statuses, were determined by calculating the mean differences (MDs), odds ratios (ORs) and p-trends adjusted for sex, age, study level and body mass index. Positive associations for IL-6 levels in the form of adjusted MDs and ORs were obtained for all PM-OP metrics, reaching statistical significance for both OP-DTT and OP-AA in the fine fraction, with adjusted OR = 5.66; 95%CI (1.46 to 21.92) and 3.32; 95%CI (1.07 to 10.35), respectively, along with statistically significant dose–response patterns when restricting to asthma and adjusted also for clinical variables (adjusted p-trend = 0.029 and 0.01). Similar or stronger associations and dose–response patterns were found for the IL-6/IL-10 ratio. In conclusion, our findings on the effect of PM-OP on systemic inflammation support that asthma is a heterogeneous disease at the molecular level, with PM-OP potentially playing an important role. Full article

Cystathionine γ-lyase (CSE) is the second enzyme in the trans-sulfuration pathway that converts cystathionine to cysteine. It is also one of three major enzymes responsible for the biosynthesis of hydrogen sulfide (H₂S). CSE is believed to be the major source of endogenous H₂S in the cardiovascular system, and the CSE/H₂S system plays a crucial role in a variety of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is less well understood, especially at the post-translational level. Here, we demonstrated that the persulfidation of CSE inhibits its activity by ~2-fold in vitro. The loss of this post-translational modification in the presence of dithiothreitol (DTT) results in a reversal of basal activity. Cys137 was identified as the site for persulfidation by combining mass spectrometry, mutagenesis, activity analysis and streptavidin–biotin pull-down assays. To test the physiological relevance of the persulfidation regulation of CSE, human aortic vascular smooth muscle cells (HA-VSMCs) were incubated with vascular endothelial growth factor (VEGF), which is known to enhance endogenous H₂S levels. Under these conditions, consistent with the change tendency of the cellular H₂S level, the CSE persulfidation levels increased transiently and then gradually decreased to the basal level. Collectively, our study revealed a negative feedback regulation mechanism of the CSE/H₂S system via the persulfidation of CSE and demonstrated the potential for maintaining cellular H₂S homeostasis under oxidative stress conditions, particularly in tissues where CSE is a major source of H₂S. Full article

Small GTP-binding proteins of the Rab family regulate intracellular vesicle trafficking across many aspects of the transport system. Among these, Rab9 is recognized for its role in controlling the transport system not only around the trans-Golgi network but also around the late endosome. However, the specific functions across different cell types and tissues remain unclear. Here, for the first time, we report that Rab9 negatively regulates morphological changes in the FBD-102b cell line, an oligodendroglial precursor cell line undergoing morphological differentiation. The knockdown of Rab9 led to an increase in cell shape alterations characterized by widespread membrane extensions. These changes were accompanied by increased expression levels of oligodendroglial cell differentiation and myelination marker proteins. Notably, the knockdown of Rab9 was capable of recovering defective cell morphological changes induced by tunicamycin, an inducer of endoplasmic reticulum (ER) stress, which is one of the major causes of oligodendroglial cell diseases such as Pelizaeus–Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]). In addition, Rab9 knockdown recovered levels of ER stress marker proteins and differentiation markers. Similar results were obtained in the cases of dithiothreitol (DTT), another chemical ER stress inducer, as well as HLD1-associated proteolipid protein 1 (PLP1) mutant protein. These results indicate a unique role for Rab9 in oligodendroglial cell morphological changes, suggesting its potential as a therapeutic target for mitigating diseases such as HLD1 at the molecular and cellular levels. Full article

Recent studies indicate that mass concentration alone is not the best parameter to assess the toxicity and the health effects of particulate matter (PM). Indeed, the chemical composition of the particles plays an important role, and oxidative potential (OP) measurements are being proposed as an alternative way to assess toxicity. The European Union (EU) is currently proposing a draft of the new air quality directive which includes OP measurements but does not specify the methods and/or protocols of analysis. In this light, the purpose of this study was to evaluate the feasibility of two literature assays, namely ascorbic acid (AA) and dithiothreitol (DTT), for routine PM analysis by testing urban PM filters from a one-year sampling campaign conducted by ARPA Lombardia. Indoor PM samples were also tested to emphasize the importance of monitoring closed spaces in which people spend most of their time. Following the optimization of the DTT assay, both methods proved to be suitable for large-scale PM analysis. The results show that the oxidative strength of urban PM is constant throughout the year for the outdoor samples (OP^AA: 0.067–0.39 nmol min⁻¹ m⁻³; OP^DTT: 0.033–0.109 nmol min⁻¹ m⁻³), indicating the need for routine OP monitoring. Instead, indoor areas were characterized by particles with a lower oxidative capacity (OP^AA: 5.40–24 pmol min⁻¹ m⁻³; OP^DTT: 9.7–32 pmol min⁻¹ m⁻³), driven both by lower concentrations and a different chemical composition. All the data collected highlight the need to add this parameter as part of the chemical characterization of PM, moving in the same direction as the new EU air quality directive. Full article

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H₂S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H₂S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits. Full article

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative TrkAIII splicing of the neurotrophin receptor tropomyosin-related kinase A (NTRK1/TrkA), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca²⁺-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca²⁺ uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca²⁺ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca²⁺-calmodulin and PI3K. This identifies Grp78, Ca²⁺-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation. Full article

This study aimed to identify the characteristics of atmospheric pollutants emitted by agricultural activities and to evaluate factors that may cause harm to human health. For the research, atmospheric pollutants were measured over the course of a year in representative rice farming and field crop farming areas in South Korea. The results confirmed that the characteristics of atmospheric pollutants in agricultural areas are influenced by the nature of agricultural activities. Specifically, when comparing rice paddies and field crop areas, during summer, the correlation between oxidative potential and levoglucosan—a marker for biomass burning—weakens due to less burning activity in the rice-growing season, leading to lower oxidative potential despite different PM_2.5 across areas. The study also finds that methyl sulfonic acid, indicating marine influence, plays a big role in keeping oxidative potential low in summer. This suggests that the main causes of PM_2.5-related health risks in the area are from biomass burning and external sources, with burning being a significant factor in increasing oxidative potential. Based on these results, it is hoped that measures can be taken in the future to reduce atmospheric pollutants in agricultural areas. Full article

Oxidation potential (OP), reflecting the redox activities of particle matter (PM), is considered an optimal measure to explain the biological effects of PM exposure. However, the size resolution of the relationship between OP and chemical composition in PM, especially how the relationship changes after respiratory exposure, has not been well investigated. In this study, size-resolved indoor PM₁₀ samples were collected from a waste recycling plant from November to December 2021 using an Anderson eight-stage cascade impactor. OP, measured by a dithiothreitol (DTT) assay (defined as OP^DTT), and elements, determined by inductively coupled plasma–mass spectrometry (ICP-MS) in size-resolved PM, were determined to check their relationships and the related human exposure risk. The results indicated that compared with PM_0.4 and PM_0.4–2.1, PM_2.1–10 contributed the most to total OP^DTT and its bound elements contributed the most to potential health risks, both before and after respiratory exposure. The association between OP^DTT and the elements varied with PM size. Pearson correlation analysis showed that the PM_0.4- and PM_0.4–2.1-bound elements were moderate-to-strongly positively correlated with OP_v^DTT (r: 0.60–0.90). No significant correlation or dose–response relationship was found in PM_2.1–10. After respiratory exposure, several PM_0.4- and PM_0.4–2.1-bound elements had a moderate-to-strongly positive correlation with deposition fluxes of OP (defined as OP^Flux) (0.69–0.90). A generalized linear model analysis showed that the interquartile range (IQR) increase in the PM-bound elements (ng h⁻¹) was associated with a 41.7–58.1% increase in OP^Flux. Our study is a special case that enriches the knowledge of the association between OP^DTT and the chemical composition of PM of different sizes, especially after respiratory exposure, but the generalizability of the findings to other settings or types of PM may be limited. The associations among OP^DTT, other chemical compositions of PM, and human exposure risk merit further research. Full article

Orthopaedic implant-associated infections (OIAIs) represent a notable complication of contemporary surgical procedures, exerting a considerable impact on patient outcomes and escalating healthcare expenditures. Prompt diagnosis holds paramount importance in managing OIAIs, with sonication widely acknowledged as the preferred method for detecting biofilm-associated infections. Recently, dithiothreitol (DTT) has emerged as a potential substitute for sonication, owing to its demonstrated ability to impede biofilm formation. This study aimed to compare the efficacy of DTT with sonication in identifying microorganisms within implants. Conducted as a prospective cohort investigation, the study encompassed two distinct groups: patients with suspected infections undergoing implant removal (Group A) and those slated for hardware explantation (Group B). Hardware segments were assessed for biofilm-related microorganisms using both sonication and DTT, with a comparative analysis of the two methods. A total of 115 patients were enrolled. In Group A, no statistically significant disparity was observed between DTT and sonication. DTT exhibited a sensitivity of 89.47% and specificity of 96.3%. Conversely, in Group B, both DTT and sonication fluid cultures yielded negative results in all patients. Consequently, this investigation suggests that DTT holds comparable efficacy to sonication in detecting OIAIs, offering a novel, cost-effective, and readily accessible diagnostic modality for identifying implant-associated infections. Full article

Hydrogels are a class of soft biomaterials and the material of choice for a myriad of biomedical applications due to their biocompatibility and highly tunable mechanical and biochemical properties. Specifically, light-mediated thiol-norbornene click reactions between norbornene-modified macromers and di-thiolated crosslinkers can be used to form base hydrogels amenable to spatial biochemical modifications via subsequent light reactions between pendant norbornenes in the hydrogel network and thiolated peptides. Macromers derived from natural sources (e.g., hyaluronic acid, gelatin, alginate) can cause off-target cell signaling, and this has motivated the use of synthetic macromers such as poly(ethylene glycol) (PEG). In this study, commercially available 8-arm norbornene-modified PEG (PEG-Nor) macromers were reacted with di-thiolated crosslinkers (dithiothreitol, DTT) to form synthetic hydrogels. By varying the PEG-Nor weight percent or DTT concentration, hydrogels with a stiffness range of 3.3 kPa–31.3 kPa were formed. Pendant norbornene groups in these hydrogels were used for secondary reactions to either increase hydrogel stiffness (by reacting with DTT) or to tether mono-thiolated peptides to the hydrogel network. Peptide functionalization has no effect on bulk hydrogel mechanics, and this confirms that mechanical and biochemical signals can be independently controlled. Using photomasks, thiolated peptides can also be photopatterned onto base hydrogels, and mesenchymal stem cells (MSCs) attach and spread on RGD-functionalized PEG-Nor hydrogels. MSCs encapsulated in PEG-Nor hydrogels are also highly viable, demonstrating the ability of this platform to form biocompatible hydrogels for 2D and 3D cell culture with user-defined mechanical and biochemical properties. Full article