In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes.

In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.

A comprehensive chemical characterization (water-soluble ions, organic and elemental carbon, water- and methanol-soluble organic carbon, levoglucosan, and major and trace metals) of PM10 samples collected in a rural area located in the southeast of the Iberian Peninsula was performed. Additionally, the oxidative potential of the samples, used as an indicator of aerosol toxicity, was determined by the ascorbic acid (OPAA) and dithiothreitol (OPDTT) assays. The average concentration of PM10 during the study period, spanning from late winter to early spring, was 20.2 ± 10.8 μg m-3. Nitrate, carbonate and calcium (accounting for 20% of the average PM10 mass concentration) and organic matter (with a contribution of 28%) were the main chemical components of PM10. Average concentrations of traffic tracers such as elemental carbon, copper and zinc (0.31 μg m-3, 3 ng m-3, and 9 ng m-3, respectively) were low compared with those obtained at an urban site in the same region, due to the almost total absence of traffic in the surrounding of the sampling site. Regarding levoglucosan and K+, which can be considered as tracers of biomass burning, their concentrations (0.12 μg m-3 and 55 ng m-3, respectively) were in the lower range of values reported for other rural areas in Europe, suggesting a moderate contribution form this source to PM10 levels. The results of the Pearson\'s correlation analysis showed that volume-normalised OPAA and OPDTT levels (average values of 0.11 and 0.32 nmol min-1 m-3, respectively) were sensitive to different PM10 chemical components. Whereas OPAA was not strongly correlated with any of the species measured, good correlation coefficients of OPDTT with water-soluble organic carbon (r = 0.81) and K+ (r = 0.73) were obtained, which points to biomass burning as an important driver of the DTT activity.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, β-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

BACKGROUND: APRI and FIB-4 scores are used to exclude clinically significant fibrosis (defined as stage ≥ F2) in patients with chronic viral hepatitis. However, the cut-offs for these scores (generated by Youden indices) vary between different patient cohorts. This study aimed to evaluate whether serum dithiothreitol-oxidizing capacity (DOC), i.e., a surrogate test of quiescin sulfhydryl oxidase-1, which is a matrix remodeling enzyme, could be used to non-invasively identify significant fibrosis in patients with various chronic liver diseases (CLDs).
METHODS: Diagnostic performance of DOC was compared with APRI and FIB-4 for identifying significant fibrosis. ROC curve analyses were undertaken in: a) two chronic hepatitis B (CHB) cohorts, independently established from hospitals in Wenzhou (n = 208) and Hefei (n = 120); b) a MASLD cohort from Wenzhou hospital (n = 122); and c) a cohort with multiple CLD etiologies (except CHB and MASLD; n = 102), which was identified from patients in both hospitals. Cut-offs were calculated using the Youden index. All CLD patients (n = 552) were then stratified by age for ROC curve analyses and cut-off calculations.
RESULTS: Stratified by CLD etiology or age, ROC curve analyses consistently showed that the DOC test was superior to APRI and FIB-4 for discriminating between clinically significant fibrosis and no fibrosis, when APRI and FIB-4 showed poor/modest diagnostic performance (P < 0.05, P < 0.01 and P < 0.001 in 3, 1 and 3 cohort comparisons, respectively). Conversely, the DOC test was equivalent to APRI and FIB-4 when all tests showed moderate/adequate diagnostic performances (P > 0.05 in 11 cohort comparisons). DOC had a significant advantage over APRI or FIB-4 scores for establishing a uniform cut-off independently of age and CLD etiology (coefficients of variation of DOC, APRI and FIB-4 cut-offs were 1.7%, 22.9% and 47.6% in cohorts stratified by CLD etiology, 2.0%, 26.7% and 29.5% in cohorts stratified by age, respectively). The uniform cut-off was 2.13, yielded from all patients examined. Surprisingly, the uniform cut-off was the same as the DOC upper limit of normal with a specificity of 99%, estimated from 275 healthy control individuals. Hence, the uniform cut-off should possess a high negative predictive value for excluding significant fibrosis in primary care settings. A high DOC cut-off with 97.5% specificity could be used for detecting significant fibrosis (≥ F2) with an acceptable positive predictive value (87.1%).
CONCLUSIONS: This proof-of-concept study suggests that the DOC test may efficiently rule out and rule in significant liver fibrosis, thereby reducing the numbers of unnecessary liver biopsies. Moreover, the DOC test may be helpful for clinicians to exclude significant liver fibrosis in the general population.

Saccharomyces cerevisiae is important for protein secretion studies, yet the complexities of protein synthesis and secretion under endoplasmic reticulum (ER) stress conditions remain not fully understood. ER stress, triggered by alterations in the ER protein folding environment, poses substantial challenges to cells, especially during heterologous protein production. In this study, we used RNA-seq to analyze the transcriptional responses of yeast strains to ER stress induced by reagents such as tunicamycin (Tm) or dithiothreitol (DTT). Our gene expression analysis revealed several crucial genes, such as HMO1 and BIO5, that are involved in ER-stress tolerance. Through metabolic engineering, the best engineered strain R23 with HMO1 overexpression and BIO5 deletion, showed enhanced ER stress tolerance and improved protein folding efficiency, leading to a 2.14-fold increase in α-amylase production under Tm treatment and a 2.04-fold increase in cell density under DTT treatment. Our findings contribute to the understanding of cellular responses to ER stress and provide a basis for further investigations into the mechanisms of ER stress at the cellular level.

An alternative metric to account for particulate matter (PM) composition-based toxicity is the ability of PM-species to generate reactive oxygen species (ROS) and deplete antioxidants, the so-called oxidative potential (OP). Acellular OP assays are the most used worldwide, mainly those based on ascorbic acid (AA) and dithiothreitol (DTT) depletion; OP values are calculated from AA/DTT concentration over time kinetic curves. Since a great variability in OP-DTT and OP-AA values can be found in the literature, the understanding of those factors affecting the kinetic rate of AA and DTT oxidation in the presence of PM-bound species will improve the interpretation of OP values. In this work, a kinetic study of the oxidation rate of AA and DTT driven by species usually found in PM (transition metals and naphthoquinone (NQ)) was carried out. In particular, the influence of the concentration of Cu(II), Fe(II), Fe(III), Mn(II), Mn(III), and 1,4-NQ, and the type of fluid used in the assay (phosphate buffer (PB), phosphate buffer saline (PBS) and artificial lysosomal fluid (ALF)) is analysed and discussed. The reaction orders with respect to the AA/DTT and the active compound, and the kinetic rate constants were also determined. The results show great variability in OP values among the studied species depending on the fluid used; the OP values were mostly higher in PB0.05 M, followed by PBS1x and ALF. Moreover, different species concentration-responses for OP-DTT/OP-AA were obtained. These differences were explained by the different reaction orders and kinetic rate constants obtained for each active compound in each fluid.

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.

The present study examined the hypothesis that changes in the oxidation-reduction state of thiol residues in functional proteins play a major role in the expression of the ventilatory responses in conscious rats that occur during a hypoxic-hypercapnic (HH) gas challenge and upon return to room air. A HH gas challenge in vehicle-treated rats elicited robust and sustained increases in minute volume (via increases in frequency of breathing and tidal volume), peak inspiratory and expiratory flows, and inspiratory and expiratory drives while minimally affecting the non-eupneic breathing index (NEBI). The HH-induced increases in these parameters, except for frequency of breathing, were substantially diminished in rats pre-treated with the potent and lipophilic disulfide-reducing agent, L,D-dithiothreitol (100 µmol/kg, IV). The ventilatory responses that occurred upon return to room air were also substantially different in dithiothreitol-treated rats. In contrast, pre-treatment with a substantially higher dose (500 µmol/kg, IV) of the lipophilic congener of the monosulfide, N-acetyl-L-cysteine methyl ester (L-NACme), only minimally affected the expression of the above-mentioned ventilatory responses that occurred during the HH gas challenge or upon return to room air. The effectiveness of dithiothreitol suggests that the oxidation of thiol residues occurs during exposure to a HH gas challenge and that this process plays an essential role in allowing for the expression of the post-HH excitatory phase in breathing. However, this interpretation is contradicted by the lack of effects of L-NACme. This apparent conundrum may be explained by the disulfide structure affording unique functional properties to dithiothreitol in comparison to monosulfides. More specifically, the disulfide structure may give dithiothreitol the ability to alter the conformational state of functional proteins while transferring electrons. It is also possible that dithiothreitol is simply a more efficient reducing agent following systemic injection, although one interpretation of the data is that the effects of dithiothreitol are not due to its reducing ability.

Atmospheric particulate matter (PM) has been related to numerous adverse health effects in humans. Nowadays, it is believed that one of the possible mechanisms of toxicity could be the oxidative stress, which involves the development of reactive oxygen species (ROS). Different assays have been proposed to characterize oxidative stress, such as dithiothreitol (DTT) and ascorbic acid (AA) acellular assays (OPDTT and OPAA), as a metric more relevant than PM mass measurement for PM toxicity. This study evaluates the OP of the bioaccessible fraction of 65 PM10 samples collected at an Atlantic Coastal European urban site using DTT and AA assays. A physiologically based extraction (PBET) using Gamble\'s solution (GS) as a simulated lung fluid (SLF) was used for the assessment of the bioaccessible fraction of PM10. The use of the bioaccessible fraction, instead of the fraction assessed using conventional phosphate buffer and ultrasounds assisted extraction (UAE), was compared for OP assessment. Correlations between OPDTT and OPAA, as well as total and bioaccessible concentrations of polycyclic aromatic hydrocarbons (PAHs) and metal(oid)s, were investigated to explore the association between those compounds and OP. A correlation was found between both OP (OPDTT and OPAA) and total and bioaccessible concentrations of PAHs and several metal(oid)s such as As, Bi, Cd, Cu, Ni, and V. Additionally, OPDTT was found to be related to the level of K+.