Widespread genomic de novo DNA methylation occurs following CD8+ T cell activation and proliferation.

This research investigates the intricate dynamics of DNA methylation in the hours following CD8+ T cell activation, during a critical yet understudied temporal window. DNA methylation is an epigenetic modification central to regulation of gene expression and directing immune responses. Our investigation spanned 96-h post-activation and unveils a nuanced tapestry of global and site-specific methylation changes. We identified 15,626 significant differentially methylated CpGs spread across the genome, with the most significant changes occurring within the genes ADAM10, ICA1, and LAPTM5. While many changes had modest effect sizes, approximately 120 CpGs exhibited a log2FC above 1.5, with cell activation and proliferation pathways the most affected. Relatively few of the differentially methylated CpGs occurred along adjacent gene regions. The exceptions were seven differentially methylated gene regions, with the Human T cell Receptor Alpha Joining Genes demonstrating consistent methylation change over a 3kb window. We also investigated whether an inflammatory environment could alter DNA methylation during activation, with proliferating cells exposed to the oxidant glycine chloramine. No substantial differential methylation was observed in this context. The temporal perspective of early activation adds depth to the evolving field of epigenetic immunology, offering insights with implications for therapeutic innovation and expanding our understanding of epigenetic modulation in immune function.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability.

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections.

Inside the phagosome: A bacterial perspective.

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.

Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress.

Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.

A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.

Faecal Myeloperoxidase as a Biomarker of Endoscopic Activity in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD], consisting of Crohn's disease [CD] and ulcerative colitis [UC], is a relapsing-remitting illness. Treat-to-target IBD management strategies require monitoring of gastrointestinal inflammation. This study aimed to investigate faecal myeloperoxidase [fMPO], a neutrophil granule enzyme, as a biomarker of IBD activity. METHODS: Prospectively recruited participants with IBD, undergoing ileocolonoscopy for disease assessment, provided biological samples and completed symptom questionnaires prior to endoscopy. fMPO, C-reactive protein [CRP], and faecal calprotectin [fCal] were compared with validated endoscopic indices [simple endoscopic score for CD and UC endoscopic index of severity]. Receiver operating characteristic [ROC] curves assessed the performance of fMPO, CRP, and fCal in predicting endoscopic disease activity. Baseline biomarkers were used to predict a composite endpoint of complicated disease at 12 months [need for escalation of biologic/immunomodulator due to relapse, steroid use, IBD-related hospitalisation, and surgery]. RESULTS: A total of 172 participants were recruited [91 female, 100 with CD]. fMPO was significantly correlated with endoscopic activity in both CD [r = 0.53, p < 0.01] and UC [r = 0.63, p < 0.01], and with fCal in all patients with IBD [r = 0.82, p < 0.01]. fMPO was effective in predicting moderate-to-severely active CD [AUROC 0.86, p < 0.01] and UC [AUROC 0.92, p < 0.01]. Individuals with a baseline fMPO > 26 µg/g were significantly more likely to reach the composite outcome at 12 months (hazard ratio [HR] 3.71, 95% confidence interval [CI] 2.07-6.64, p < 0.01). CONCLUSIONS: Faecal myeloperoxidase is an accurate biomarker of endoscopic activity in IBD and predicted a more complicated IBD course during follow-up.

Oxidation of bacillithiol during killing of Staphylococcus aureus USA300 inside neutrophil phagosomes.

Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low-molecular-weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH-deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase-derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms.

Hypothiocyanous Acid Disrupts the Barrier Function of Brain Endothelial Cells.

Inflammation is a common feature of neurological diseases. During neuroinflammation, neutrophils are recruited to the brain vasculature, where myeloperoxidase can produce hypochlorous acid and the less well-studied oxidant hypothiocyanous acid (HOSCN). In this study, we exposed primary brain endothelial cells (BECs) to HOSCN and observed a rapid loss of transendothelial electrical resistance (TEER) at sublethal concentrations. Decreased barrier function was associated with a loss of tight junctions at cellular contacts and a concomitant loss of dynamic microtubules. Both tight junction and cytoskeletal disruptions were visible within 30 min of exposure, whereas significant loss of TEER took more than 1 h. The removal of the HOSCN after 30 min prevented subsequent barrier dysfunction. These results indicate that BECs are sensitive to HOSCN, resulting in the eventual loss of barrier function. We hypothesise that this mechanism may be relevant in neutrophil transmigration, with HOSCN facilitating blood-brain barrier opening at the sites of egress. Furthermore, this mechanism may be a way through which neutrophils, residing in the vasculature, can influence neuroinflammation in diseases.

Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer's disease.

INTRODUCTION: Neutrophil accumulation is a well-established feature of Alzheimer's disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. MATERIALS AND METHODS: We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. RESULTS: There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. CONCLUSION: Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.

Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision.

SignificanceTo move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors.

Resistance of Streptococcus pneumoniae to Hypothiocyanous Acid Generated by Host Peroxidases.

Streptococcus pneumoniae is a serious human respiratory pathogen. It generates hydrogen peroxide (H2O2) as part of its normal metabolism, yet it lacks enzymes that remove this oxidant. Here we show that lactoperoxidase and myeloperoxidase, two host enzymes present in the respiratory tract, convert bacterial H2O2 into HOSCN that S. pneumoniae can resist. We found that incubation of S. pneumoniae with myeloperoxidase in chloride-rich buffer killed the bacteria due to formation of toxic hypochlorous acid (HOCl). However, the addition of physiological concentrations of thiocyanate protected the bacteria. Similarly, S. pneumoniae remained viable in the presence of lactoperoxidase and thiocyanate even though the majority of bacterial H2O2 was converted to hypothiocyanous acid (HOSCN). S. pneumoniae and Pseudomonas aeruginosa, another respiratory pathogen, were similarly sensitive to H2O2 and HOCl. In contrast, S. pneumoniae tolerated much higher doses of HOSCN than P. aeruginosa. When associated with neutrophil extracellular traps (NETs), S. pneumoniae continued to generate H2O2, which was converted to HOCl by myeloperoxidase (MPO) present on NETs. However, there was no loss in bacterial viability because HOCl was scavenged by the NET proteins. We conclude that at sites of infection, bacteria will be protected from HOCl by thiocyanate and extracellular proteins including those associated with NETs. Resistance to HOSCN may give S. pneumoniae a survival advantage over other pathogenic bacteria. Understanding the mechanisms by which S. pneumoniae protects itself from HOSCN may reveal novel strategies for limiting the colonization and pathogenicity of this deadly pathogen.

Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates large amounts of hydrogen peroxide (H2O2), which will be converted to hypothiocyanous acid (HOSCN) by lactoperoxidase (LPO) in the human respiratory tract. S. pneumoniae has been shown to be more resistant to HOSCN than some bacteria, and sensitizing S. pneumoniae to HOSCN may be a novel treatment strategy for combating this deadly pathogen. In this study we investigated the role of the low molecular weight thiol glutathione in HOSCN resistance. S. pneumoniae does not synthesize glutathione but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidized in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione (ΔgshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H2O2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δgor). This research highlights the importance of glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilization by S. pneumoniae may be a way to limit colonization and pathogenicity.

Insights into H2O2-induced signaling in Jurkat cells from analysis of gene expression.

Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2O2-dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signaling.

Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity.

Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), Pseudomonas aeruginosa (PAO1), Staphylococcus aureus and Candida albicans, and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition. Notably, DPI added up to 60-90 min after stimulation still gave significant inhibition of subsequent NET formation. As most of the NOX2 activity had already occurred by that time, this indicates a requirement for late-stage low-level oxidant production. Inhibition of histone citrullination did not suppress NET formation, indicating that this was not the essential oxidant-dependent step. With PMA and P. aeruginosa PAO1, MPO activity played an important role in the induction of NETs and MPO inhibitors added up to 30-90 min after stimulation suppressed NET formation. NET formation with S. aureus and C. albicans was insensitive to MPO inhibition. Thus, MPO products are important with some stimuli but not others. Our results extend earlier observations with PMA and show that induction of NETs by microbial stimuli requires late stage oxidant production. Others have shown that NET formation involves NOX2-dependent elastase release from granules. As this is an early event, we conclude from our results that there is more than one oxidant-dependent step.

Ascorbate Inhibits Proliferation and Promotes Myeloid Differentiation in TP53-Mutant Leukemia.

Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53. Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1Met, a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1Met-induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1Met.

Patient understanding of and participation in infection-related care across surgical pathways: a scoping review.

OBJECTIVE: To explore the existing evidence on patient understanding of and/or participation in infection-related care in surgical specialties. METHOD: A scoping review of the literature was conducted. PubMed, Web of Science, Scopus, and grey literature sources were searched using predefined search criteria for policies, guidelines, and studies in the English language. Data synthesis was done through content and thematic analysis to identify key themes in the included studies. RESULTS: The initial search identified 604 studies, of which 41 (36 from high-income and five from low- and middle-income countries) were included in the final review. Most of the included studies focused on measures to engage patients in infection prevention and control (IPC) activities, with few examples of antimicrobial stewardship (AMS) engagement strategies. While patient engagement interventions in infection-related care varied depending on study goals, surgical wound management was the most common intervention. AMS engagement was primarily limited to needs assessment, without follow-up to address such needs. CONCLUSION: Existing evidence highlights a gap in patient participation in infection-related care in the surgical pathway. Standardization of patient engagement strategies is challenging, particularly in the context of surgery, where several factors influence how the patient can engage and retain information. Infection-related patient engagement and participation strategies in surgery need to be inclusive and contextually fit.

Peroxiredoxin 2 oxidation reveals hydrogen peroxide generation within erythrocytes during high-dose vitamin C administration.

Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H2O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 µM H2O2/min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5-3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation.