Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Sleep deprivation is highly prevalent in the modern world, possibly reaching epidemic proportions. While multiple theories regarding the roles of sleep exist (inactivity, energy conservation, restoration, brain plasticity and antioxidant), multiple unknowns still remain regarding the proposed antioxidant roles of sleep. The existing experimental evidence is often contradicting, with studies pointing both toward and against the presence of oxidative stress after sleep deprivation. The main goals of this review were to analyze the existing experimental data regarding the relationship between sleep deprivation and oxidative stress, to attempt to further clarify multiple aspects surrounding this relationship and to identify current knowledge gaps. Systematic searches were conducted in three major online databases for experimental studies performed on rat models with oxidative stress measurements, published between 2015 and 2022. A total of 54 studies were included in the review. Most results seem to point to changes in oxidative stress parameters after sleep deprivation, further suggesting an antioxidant role of sleep. Alterations in these parameters were observed in both paradoxical and total sleep deprivation protocols and in multiple rat strains. Furthermore, the effects of sleep deprivation seem to extend beyond the central nervous system, affecting multiple other body sites in the periphery. Sleep recovery seems to be characterized by an increased variability, with the presence of both normalizations in some parameters and long-lasting changes after sleep deprivation. Surprisingly, most studies revealed the presence of a stress response following sleep deprivation. However, the origin and the impact of the stress response during sleep deprivation remain somewhat unclear. While a definitive exclusion of the influence of the sleep deprivation protocol on the stress response is not possible, the available data seem to suggest that the observed stress response may be determined by sleep deprivation itself as opposed to the experimental conditions. Due to this fact, the observed oxidative changes could be attributed directly to sleep deprivation.

Gamma-glutamyl transferase 1 (GGT1) is a critical enzyme involved in the hydrolysis and/or transfer of gamma-glutamyl groups of glutathione, which helps maintain cysteine levels in plasma. In this study, we synthesized L-ABBA analogs to investigate their inhibitory effect on GGT1 hydrolysis and transpeptidase activity, with the goal of defining the pharmacophore of L-ABBA. Our structure-activity relationship (SAR) study revealed that an α-COO- and α-NH3+ group, as well as a two-CH2 unit distance between α-C and boronic acid, are essential for the activity. The addition of an R (alkyl) group at the α-C reduced the activity of GGT1 inhibition, with L-ABBA being the most potent inhibitor among the analogs. Next, we investigated the impact of L-ABBA on plasma levels of cysteine and GSH species, with the expectation of observing reduced cysteine levels and enhanced GSH levels due to its GGT1 inhibition. We administered L-ABBA intraperitoneally and determined the plasma levels of cysteine, cystine, GSH, and GSSG using LCMS. Our results showed time- and dose-dependent L-ABBA changes in total plasma cysteine and GSH levels. This study is the first to demonstrate the regulation of plasma thiol species upon GGT1 inhibition, with plasma cystine levels reduced by up to ∼ 75 % with L-ABBA (0.3 mg/dose). Cancer cells are highly dependent on the uptake of cysteine from plasma for maintaining high levels of intracellular glutathione. Thus, our findings suggest that GGT1 inhibitors, such as L-ABBA, have the potential to be used in GSH reduction thereby inducing oxidative stress in cancer cells and reducing their resistance to many chemotherapeutic agents.

Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen-glucose deprivation (OGD)-induced human umbilical endothelial cells\' (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the levels of the glutamate-cysteine ligase catalytic subunit (GCLc) and the glutathione synthetase (GSS) proteins, a key enzyme for de novo GSH synthesis, were determined using enzyme-linked immunoassay (ELISA). We demonstrated that the SDX treatment in OGD conditions significantly elevated the intracellular GSH, enhanced the GSH:GSSG ratio, shifting the redox potential to a more pro-reducing status. Furthermore, SDX increased the levels of both GCLc and GSS. The results show that SDX protects the human endothelial cells against ischemic stress by affecting the GSH levels and cellular redox state. These changes suggest that the reduction in the ischemia-induced vascular endothelial cell injury through repressing apoptosis and oxidative stress associated with SDX treatment may be due to an increase in GSH synthesis and modulation of the GSH redox system.

Asthma is associated with oxidative stress and oxidative damage of biomolecules, including DNA. Here, we describe the protocols to quantify reactive oxygen species (ROS) and oxidative stress markers in a mouse model of allergic airway inflammation. We also provide detailed methods to measure DNA damage by long-run real-time PCR for DNA-damage quantification (LORD-Q) assay and gene-specific DNA damage analyses by long amplicon (LA)-qPCR. Additionally, we describe methods to quantify oxidized DNA base lesions in lung genomic DNA by mass spectrometry, and to measure enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1). Using these methods, the levels of oxidative stress and DNA damage in allergic inflammation and asthma can be elucidated.

The COVID-19 pandemic has been inflicted upon humanity by the SARS-CoV-2 virus, the latest insidious incarnation of the coronaviruses group. While in its wake intense scientific research has produced breakthrough vaccines and cures, there still exists an immediate need to further understand the origin, mechanobiology and biochemistry, and destiny of this virus so that future pandemics arising from similar coronaviruses may be contained more effectively. In this Perspective, we discuss the various evidential findings of virus propagation and connect them to respective underpinning cellular biomechanical states leading to corresponding manifestations of the viral activity. We further propose avenues to tackle the virus, including from a \"musical\" vantage point, and contain its relentless strides that are currently afflicting the global populace.

Elemental sulfur is a common fungicide, but its inhibition mechanism is unclear. Here, we investigated the effects of elemental sulfur on the single-celled fungus Saccharomyces cerevisiae and showed that the inhibition was due to its function as a strong oxidant. It rapidly entered S. cerevisiae. Inside the cytoplasm, it reacted with glutathione to generate glutathione persulfide that then reacted with another glutathione to produce H2S and glutathione disulfide. H2S reversibly inhibited the oxygen consumption by the mitochondrial electron transport chain, and the accumulation of glutathione disulfide caused disulfide stress and increased reactive oxygen species in S. cerevisiae. Elemental sulfur inhibited the growth of S. cerevisiae; however, it did not kill the yeast for up to 2 h exposure. The combined action of elemental sulfur and hosts\' immune responses may lead to the demise of fungal pathogens.

Oilseed rape (Brassica napus) is an economically important crop. In a temperate climate, powdery mildew Erysiphe crucifertaum can drastically reduce its yield. Nevertheless, cultivars resistant to this fungal disease have not yet been selected. Glutathione S-transferase GSTF11 is involved in glucosinolate (GSL) biosynthesis and response to stress, including fungal deceases. However, the impact of exogenous GSTF11 gene expression on resistance to powdery mildew has not yet been confirmed and requires further investigation. Transgenic B. napus was generated for this purpose. It demonstrated increased GST activity and a higher GSH:GSSG ratio under normal conditions. Powdery mildew Erysiphe crucifertaum caused 50% mortality in wild type (WT) plants. In most of transgenic plants, mycelium growth was inhibited. The infection contributed to higher GSTF11 expression and increased levels of glutathione (GSH) and oxidized glutathione (GSSG) in both transgenic and WT plants. In contrast, GSTF11 mRNA content, GST activity and GSSG level were lower only in WT plants. In transgenic plants, increased resistance to powdery mildew correlated with a lower GSH:GSSG ratio, indicating a higher content of neutralized toxic molecules. GSTF11 expression was also affected by cold stress, but not drought. At -1 °C, the expression level increased only in transgenic plants. Therefore, GSTF11 appears to be nonspecific and is able to protect plants under several types of stress. This gene could be used as a target in the production of stress tolerant cultivars.

Purpose: This study aimed to assess the requirement of protein in pre-exercise carbohydrate drinks for optimal endurance performance at high intensity and post-exercise fatigue recovery. Methods: Endurance performance at 85% V . ⁢ O 2 peak of young men (age 20 ± 0.9 years, V . ⁢ 2peak 49.3 ± 0.3 L/min) was measured for two consecutive days using cycling time to exhaustion and total work exerted 2 h after three isocaloric supplementations: RICE (50 g, protein: 1.8 g), n = 7; SOY + RICE (50 g, protein: 4.8 g), n = 7; and WHEY + RICE (50 g, protein: 9.2 g), n = 7. Results: Endurance performance was similar for the three supplemented conditions. Nevertheless, maximal cycling time and total exerted work from Day 1 to Day 2 were improved in the WHEY + RICE (+21%, p = 0.05) and SOY-RICE (+16%, p = 0.10) supplemented conditions, not the RICE supplemented condition. Increases in plasma interleukin-6 (IL-6) were observed 1 h after exercise regardless of supplemented conditions. Plasma creatine kinase remained unchanged after exercise for all three supplemented conditions. Increases in ferric reducing antioxidant power (FRAP) after exercise were small and similar for the three supplemented conditions. Conclusion: Adding protein into carbohydrate drinks provides no immediate benefit in endurance performance and antioxidant capacity yet enhances fatigue recovery for the next day. Soy-containing carbohydrate drink, despite 50% less protein content, shows similar fatigue recovery efficacy to the whey protein-containing carbohydrate drink. These results suggest the importance of dietary nitrogen sources in fatigue recovery after exercise.

In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell weight) are often used. Nonetheless, detailed procedures for calculation of these production yields are often omitted in research articles, whereas methods for product quantification are described well. Here, we describe a detailed calculation procedure from our previous studies on glutathione production by Saccharomyces cerevisiae. This procedure can be applied to various other products and microorganisms, and therefore, may prove to be useful in various other bioproduction studies.