Ferroptosis in health and disease.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

The 'PSILAUT' protocol: an experimental medicine study of autistic differences in the function of brain serotonin targets of psilocybin.

BACKGROUND: The underlying neurobiology of the complex autism phenotype remains obscure, although accumulating evidence implicates the serotonin system and especially the 5HT2A receptor. However, previous research has largely relied upon association or correlation studies to link differences in serotonin targets to autism. To directly establish that serotonergic signalling is involved in a candidate brain function our approach is to change it and observe a shift in that function. We will use psilocybin as a pharmacological probe of the serotonin system in vivo. We will directly test the hypothesis that serotonergic targets of psilocybin - principally, but not exclusively, 5HT2A receptor pathways-function differently in autistic and non-autistic adults. METHODS: The 'PSILAUT' "shiftability" study is a case-control study autistic and non-autistic adults. How neural responses 'shift' in response to low doses (2 mg and 5 mg) of psilocybin compared to placebo will be examined using multimodal techniques including functional MRI and EEG. Each participant will attend on up to three separate visits with drug or placebo administration in a double-blind and randomized order. RESULTS: This study will provide the first direct evidence that the serotonin targets of psilocybin function differently in the autistic and non-autistic brain. We will also examine individual differences in serotonin system function. CONCLUSIONS: This work will inform our understanding of the neurobiology of autism as well as decisions about future clinical trials of psilocybin and/or related compounds including stratification approaches. TRIAL REGISTRATION: NCT05651126.

Ultralong transients enhance sensitivity and resolution in Orbitrap-based single-ion mass spectrometry.

Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.

Expanding Differential Ion Mobility Separations into the MegaDalton Range.

Along with mass spectrometry (MS), ion mobility separations (IMS) are advancing to ever larger biomolecules. The emergence of electrospray ionization (ESI) and native MS enabled the IMS/MS analyses of proteins up to ∼100 kDa in the 1990s and whole protein complexes and viruses up to ∼10 MDa since the 2000s. Differential IMS (FAIMS) is substantially orthogonal to linear IMS based on absolute mobility K and offers exceptional resolution, unique selectivity, and steady filtering readily compatible with slower analytical methods such as electron capture or transfer dissociation (ECD/ETD). However, the associated MS stages had limited FAIMS to ions with m/z < 8000 and masses under ∼300 kDa. Here, we integrate high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer that can handle m/z up to 80,000 and MDa-size ions in the native ESI regime. In the initial evaluation, the oligomers of monoclonal antibody adalimumab (148 kDa) are size-selected up to at least the nonamers (1.34 MDa) with m/z values up to ∼17,000. This demonstrates the survival and efficient separation of noncovalent MDa assemblies in the FAIMS process, opening the door to novel analyses of the heaviest macromolecules.

Using the heme peroxidase APEX2 to probe intracellular H2O2 flux and diffusion.

Currently available genetically encoded H2O2 probes report on the thiol redox state of the probe, which means that they reflect the balance between probe thiol oxidation and reduction. Here we introduce the use of the engineered heme peroxidase APEX2 as a thiol-independent chemogenetic H2O2 probe that directly and irreversibly converts H2O2 molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, specificity, and the ability to quantitate endogenous H2O2 turnover. We show how the probe can be used to detect changes in endogenous H2O2 generation and to assess the roles and relative contributions of endogenous H2O2 scavengers. Furthermore, APEX2 can be used to study H2O2 diffusion inside the cytosol. Finally, APEX2 reveals the impact of commonly used alkylating agents and cell lysis protocols on cellular H2O2 generation.

Editorial Perspective: Bridging the translational neuroscience gap in autism - development of the 'shiftability' paradigm.

Clinical trials of pharmacological candidates targeting the core features of autism have largely failed. This is despite evidence linking differences in multiple neurochemical systems to brain function in autism. While this has in part been explained by the heterogeneity of the autistic population, the field has largely relied upon association studies to link brain chemistry to function. The only way to directly establish that a neurotransmitter or neuromodulator is involved in a candidate brain function is to change it and observe a shift in that function. This experimental approach dominates preclinical neuroscience, but not human studies. There is little direct experimental evidence describing how neurochemical systems modulate information processing in the living human brain. Thus, our understanding of how neurochemical differences contribute to neurodiversity is limited, impeding our ability to translate findings from animal studies into humans. Here, we introduce our 'shiftability' paradigm, an approach to bridge the translational gap in autism research. We provide an overview of the guiding principles and methodologies we use to directly test the hypothesis that neurochemical systems function differently in autistic and non-autistic individuals.

Zika virus remodelled ER membranes contain proviral factors involved in redox and methylation pathways.

Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes. Here, we isolate ER membranes from ZIKV-infected cells and determine their proteome. Forty-six host cell factors are enriched in ZIKV remodeled membranes, several of these having a role in redox and methylation pathways. Four proteins are characterized in detail: thioredoxin reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and increasing stability of viral transmembrane proteins; adenosylhomocysteinase (AHCY) indirectly promoting m6A methylation of ZIKV RNA by decreasing the level of S- adenosyl homocysteine and thus, immune evasion. These results highlight the involvement of redox and methylation enzymes in the ZIKV life cycle and their accumulation at virally remodeled ER membranes.

Proteome-wide tagging with an H2O2 biosensor reveals highly localized and dynamic redox microenvironments.

Hydrogen peroxide (H2O2) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H2O2 can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H2O2 generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H2O2 sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H2O2-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H2O2 availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H2O2 generation is much more localized and functionally differentiated than previously recognized.

Treatment and Survival Among Patients With Colorectal Cancer in Sub-Saharan Africa: A Multicentric Population-Based Follow-Up Study.

BACKGROUND: The burden of colorectal cancer (CRC) is increasing in Sub-Saharan Africa (SSA). However, little is known about CRC treatment and survival in the region. METHODS: A random sample of 653 patients with CRC diagnosed from 2011 to 2015 was obtained from 11 population-based cancer registries in SSA. Information on clinical characteristics, treatment, and/or vital status was obtained from medical records in treating hospitals for 356 (54%) of the patients ("traced cohort"). Concordance of CRC treatment with NCCN Harmonized Guidelines for SSA was assessed. A Cox proportional hazards model was used to examine the association between survival and human development index (HDI). RESULTS: Of the 356 traced patients with CRC, 51.7% were male, 52.8% were from countries with a low HDI, 55.1% had colon cancer, and 73.6% were diagnosed with nonmetastatic (M0) disease. Among the patients with M0 disease, however, only 3.1% received guideline-concordant treatment, 20.6% received treatment with minor deviations, 31.7% received treatment with major deviations, and 35.1% received no treatment. The risk of death in patients who received no cancer-directed therapy was 3.49 (95% CI, 1.83-6.66) times higher than in patients who received standard treatment or treatment with minor deviations. Similarly, the risk of death in patients from countries with a low HDI was 1.67 (95% CI, 1.07-2.62) times higher than in those from countries with a medium HDI. Overall survival at 1 and 3 years was 70.9% (95% CI, 65.5%-76.3%) and 45.3% (95% CI, 38.9%-51.7%), respectively. CONCLUSIONS: Fewer than 1 in 20 patients diagnosed with potentially curable CRC received standard of care in SSA, reinforcing the need to improve healthcare infrastructure, including the oncology and surgical workforce.

Methane formation driven by light and heat prior to the origin of life and beyond.

Methane is a potent greenhouse gas, which likely enabled the evolution of life by keeping the early Earth warm. Here, we demonstrate routes towards abiotic methane and ethane formation under early-earth conditions from methylated sulfur and nitrogen compounds with prebiotic origin. These compounds are demethylated in Fenton reactions governed by ferrous iron and reactive oxygen species (ROS) produced by light and heat in aqueous environments. After the emergence of life, this phenomenon would have greatly intensified in the anoxic Archean by providing methylated sulfur and nitrogen substrates. This ROS-driven Fenton chemistry can occur delocalized from serpentinization across Earth's humid realm and thereby substantially differs from previously suggested methane formation routes that are spatially restricted. Here, we report that Fenton reactions driven by light and heat release methane and ethane and might have shaped the chemical evolution of the atmosphere prior to the origin of life and beyond.

The influence of alkylating agents on sulfur-sulfur bonds in per- and polysulfides.

Per- and polysulfides are sulfane sulfur species produced inside living cells, in organisms as diverse as bacteria, plants and humans, but their biological roles remain to be fully understood. Unfortunately, due to their reactivity, per- and polysulfides are easily altered, interconverted or lost during the processing and analysis of biological material. Thus, all current analytical methods make use of alkylating agents, to quench reactivity of hydropersulfides and hydropolysulfides and also to prevent free thiols from attacking sulfur chains in hydropolysulfides and dialkyl polysulfides. However, recent findings reveal that alkylating agents can also destroy per- and polysulfides, to varying degrees, depending on the choice of alkylating agent. Here, we discuss the challenges associated with the alkylation of per- and polysulfides, the single most important step for their preservation and detection in biological samples.

Performance of a Support Vector Machine Learning Tool for Diagnosing Diabetic Retinopathy in Clinical Practice.

PURPOSE: To examine the real-world performance of a support vector machine learning software (RetinaLyze) in order to identify the possible presence of diabetic retinopathy (DR) in patients with diabetes via software implementation in clinical practice. METHODS: 1001 eyes from 1001 patients-one eye per patient-participating in the Danish National Screening Programme were included. Three independent ophthalmologists graded all eyes according to the International Clinical Diabetic Retinopathy Disease Severity Scale with the exact level of disease being determined by majority decision. The software detected DR and no DR and was compared to the ophthalmologists' gradings. RESULTS: At a clinical chosen threshold, the software showed a sensitivity, specificity, positive predictive value and negative predictive value of 84.9% (95% CI: 81.8-87.9), 89.9% (95% CI: 86.8-92.7), 92.1% (95% CI: 89.7-94.4), and 81.0% (95% CI: 77.2-84.7), respectively, when compared to human grading. The results from the routine screening were 87.0% (95% CI: 84.2-89.7), 85.3% (95% CI: 81.8-88.6), 89.2% (95% CI: 86.3-91.7), and 82.5% (95% CI: 78.5-86.0), respectively. AUC was 93.4%. The reference graders Conger's Exact Kappa was 0.827. CONCLUSION: The software performed similarly to routine grading with overlapping confidence intervals, indicating comparable performance between the two groups. The intergrader agreement was satisfactory. However, evaluating the updated software alongside updated clinical procedures is crucial. It is therefore recommended that further clinical testing before implementation of the software as a decision support tool is conducted.

A cohort follow-up study for diabetic retinopathy screening incidence in the North Denmark Region.

AIMS: To evaluate diabetic retinopathy (DR) screening incidence in a universal healthcare system. METHODS: Registry-based cohort study based on a Danish regional population from 2009 to 2018. Individuals with diabetes were identified by medication. Screening attendance was estimated by surrogate measures using local and nationwide databases reported by cumulative incidence. RESULTS: 18,832 patients were included. By the end of the first year, the cumulative incidence of screening for DR was 60.2% and by the end of the second year 74.2%. The cumulative incidence was 93.9% overall, 97.7% for patients with type 1 diabetes (T1D) and 93.4% for patients with type 2 diabetes. Screening proportions per 1, 2 and 5 years were calculated. Females, patients with T1D, and patients attending screening at hospitals had a higher Hazard Ratio of 1.084, 1.157, and 1.573, respectively. The Cochran-Armitage trend test indicated increased screening frequency from 2009 to 2018. Validation of DR screening was done at hospitals with a mean positive predictive value of 86.78%. Cumulative incidence curves showed a small right shift when censoring the first, second and third screening visits. CONCLUSIONS: Nearly all patients were screened for DR over a 5-year timespan. Female patients with T1D who attended screening at hospitals were significantly more likely to be screened. Validation of screening visits at hospitals was reported with a high mean positive predictive value. Most other studies, to the best of our knowledge, only report screening attendance for patients already enrolled in a DR screening programme. This study describes the overall screening attendance for the total eligible diabetes population.

Tracking carbon from subduction to outgassing along the Aleutian-Alaska Volcanic Arc.

Subduction transports volatiles between Earth's mantle, crust, and atmosphere, ultimately creating a habitable Earth. We use isotopes to track carbon from subduction to outgassing along the Aleutian-Alaska Arc. We find substantial along-strike variations in the isotopic composition of volcanic gases, explained by different recycling efficiencies of subducting carbon to the atmosphere via arc volcanism and modulated by subduction character. Fast and cool subduction facilitates recycling of ~43 to 61% sediment-derived organic carbon to the atmosphere through degassing of central Aleutian volcanoes, while slow and warm subduction favors forearc sediment removal, leading to recycling of ~6 to 9% altered oceanic crust carbon to the atmosphere through degassing of western Aleutian volcanoes. These results indicate that less carbon is returned to the deep mantle than previously thought and that subducting organic carbon is not a reliable atmospheric carbon sink over subduction time scales.

Emergence of (hydro)persulfides as suppressors of lipid peroxidation and ferroptotic cell death.

Recognition of the prevalence of hydropersulfides (RSSH) and characterization of their enhanced two-electron reactivity relative to thiols have led to their implication in maintaining cellular redox homeostasis, in addition to other potential roles. Recent attention on the one-electron reactivity of RSSH has uncovered their potent radical-trapping antioxidant activity, which enables them to inhibit phospholipid peroxidation and associated cell death by ferroptosis. Herein, we briefly review key aspects of the reactivity and underlying physicochemical properties of RSSH. We emphasize their reactivity to radicals-particularly lipid peroxyl radicals that propagate the lipid peroxidation chain reaction-and the recent recognition that this results in ferroptosis suppression. We highlight open questions related to recent developments in this area and, given that all living organisms possess the ability to synthesize persulfides endogenously, suggest they may be primordial radical scavengers that occurred early in evolution and still play a role today.

Opening a window to the acutely injured brain: Simultaneous retinal and cerebral vascular monitoring in rats.

Many recent research projects have described typical chronic changes in the retinal vasculature for diverse neurovascular and neurodegenerative disorders such as stroke or Alzheimer's disease. Unlike cerebral vasculature, retinal blood vessels can be assessed non-invasively by retinal vessel analysis. To date, there is only a little information about potential simultaneous reactions of retinal and cerebral vessels in acute neurovascular diseases. The field of applications of retinal assessment could significantly be widened if more information about potential correlations between those two vascular beds and the feasibility of non-invasive retinal vessel analysis in acute neurovascular disease were available. Here, we present our protocol for the simultaneous assessment of retinal and cerebral vessels in an acute setting in anesthetized rats using a non-invasive retinal vessel analyzer and a superficial tissue imaging system for laser speckle contrast analysis via a closed bone window. We describe the experimental set-up in detail, outline the pitfalls of repeated retinal vessel analyses in an experimental set-up of several hours, and address issues that arise from the simultaneous use of two different assessment tools. Finally, we demonstrate the robustness and variability of the reactivity of retinal vessels to hypercapnia at baseline as well as their reproducibility over time using two anesthetic protocols common for neurovascular research. In summary, the procedures described in this protocol allow us to directly compare retinal and cerebral vascular beds and help to substantiate the role of the retina as a "window to the brain."

DOPA Residues Endow Collagen with Radical Scavenging Capacity.

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.

The GAPDH redox switch safeguards reductive capacity and enables survival of stressed tumour cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response to hydrogen peroxide, leading to rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable to stimulate the oxidative pentose phosphate pathway and enhance their reductive capacity. Specifically, we find that anchorage-independent growth of cells and spheroids is limited by an elevation of endogenous peroxide levels and is largely dependent on a functional GAPDH redox switch. Likewise, tumour growth in vivo is limited by peroxide stress and suppressed when the GAPDH redox switch is disabled in tumour cells. The induction of additional intratumoural oxidative stress by chemo- or radiotherapy synergized with the deactivation of the GAPDH redox switch. Mice lacking the GAPDH redox switch exhibit altered fatty acid metabolism in kidney and heart, apparently in compensation for the lack of the redox switch. Together, our findings demonstrate the physiological and pathophysiological relevance of oxidative GAPDH inactivation in mammals.

Self-supervised attention-based deep learning for pan-cancer mutation prediction from histopathology.

The histopathological phenotype of tumors reflects the underlying genetic makeup. Deep learning can predict genetic alterations from pathology slides, but it is unclear how well these predictions generalize to external datasets. We performed a systematic study on Deep-Learning-based prediction of genetic alterations from histology, using two large datasets of multiple tumor types. We show that an analysis pipeline that integrates self-supervised feature extraction and attention-based multiple instance learning achieves a robust predictability and generalizability.

3-Mercaptopyruvate sulfur transferase is a protein persulfidase.

Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H2S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.