TET1 displays catalytic and non-catalytic functions in the adult mouse cortex.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis.

Monocytes can develop an exhausted memory state characterized by reduced differentiation, pathogenic inflammation, and immune suppression that drives immune dysregulation during sepsis. Chromatin alterations, notably via histone modifications, underlie innate immune memory, but the contribution of DNA methylation remains poorly understood. Using an ex vivo sepsis model, we show altered DNA methylation throughout the genome of exhausted monocytes, including genes implicated in immune dysregulation during sepsis and COVID-19 infection (e.g., Plac8). These changes are recapitulated in septic mice induced by cecal slurry injection. Methylation profiles developed in septic mice are maintained during ex vivo culture, supporting the involvement of DNA methylation in stable monocyte exhaustion memory. Methylome reprogramming is driven in part by Wnt signaling inhibition in exhausted monocytes and can be reversed with DNA methyltransferase inhibitors, Wnt agonists, or immune training molecules. Our study demonstrates the significance of altered DNA methylation in the maintenance of stable monocyte exhaustion memory.

Epigenetic regulation of innate immune dynamics during inflammation.

Innate immune cells play essential roles in modulating both immune defense and inflammation by expressing a diverse array of cytokines and inflammatory mediators, phagocytizing pathogens to promote immune clearance, and assisting with the adaptive immune processes through antigen presentation. Rudimentary innate immune "memory" states such as training, tolerance, and exhaustion develop based on the nature, strength, and duration of immune challenge, thereby enabling dynamic transcriptional reprogramming to alter present and future cell behavior. Underlying transcriptional reprogramming are broad changes to the epigenome, or chromatin alterations above the level of DNA sequence. These changes include direct modification of DNA through cytosine methylation as well as indirect modifications through alterations to histones that comprise the protein core of nucleosomes. In this review, we will discuss recent advances in our understanding of how these epigenetic changes influence the dynamic behavior of the innate immune system during both acute and chronic inflammation, as well as how stable changes to the epigenome result in long-term alterations of innate cell behavior related to pathophysiology.

Alleviation of monocyte exhaustion by BCG derivative mycolic acid.

Monocyte exhaustion with sustained pathogenic inflammation and immune-suppression, a hallmark of sepsis resulting from systemic infections, presents a challenge with limited therapeutic solutions. This study identified Methoxy-Mycolic Acid (M-MA), a branched mycolic acid derived from Mycobacterium bovis Bacillus Calmette-Guérin (BCG), as a potent agent in alleviating monocyte exhaustion and restoring immune homeostasis. Co-treatment of monocytes with M-MA effectively blocked the expansion of Ly6Chi/CD38hi/PD-L1hi monocytes induced by LPS challenges and restored the expression of immune-enhancing CD86. M-MA treatment restored mitochondrial functions of exhausted monocytes and alleviated their suppressive activities on co-cultured T cells. Independent of TREM2, M-MA blocks Src-STAT1-mediated inflammatory polarization and reduces the production of immune suppressors TAX1BP1 and PLAC8. Whole genome methylation analyses revealed M-MA's ability to erase the methylation memory of exhausted monocytes, particularly restoring Plac8 methylation. Together, our data suggest M-MA as an effective agent in restoring monocyte homeostasis with a therapeutic potential for treating sepsis.

Monocytes reprogrammed by 4-PBA potently contribute to the resolution of inflammation and atherosclerosis.

Background: Chronic inflammation initiated by inflammatory monocytes underlies the pathogenesis of atherosclerosis. However, approaches that can effectively resolve chronic low-grade inflammation targeting monocytes are not readily available. The small chemical compound 4-phenylbutyric acid (4-PBA) exhibits broad anti-inflammatory effects in reducing atherosclerosis. Selective delivery of 4-PBA reprogrammed monocytes may hold novel potential in providing targeted and precision therapeutics for the treatment of atherosclerosis. Methods: Systems analyses integrating single-cell RNA-sequencing and complementary immunological approaches characterized key resolving characteristics as well as defining markers of reprogrammed monocytes trained by 4-PBA. Molecular mechanisms responsible for monocyte reprogramming was assessed by integrated biochemical and genetic approaches. The inter-cellular propagation of homeostasis resolution was evaluated by co-culture assays with donor monocytes trained by 4-PBA and recipient naïve monocytes. The in vivo effects of monocyte resolution and atherosclerosis prevention by 4-PBA were assessed with the high fat diet-fed ApoE -/- mouse model with i.p. 4-PBA administration. Furthermore, the selective efficacy of 4-PBA trained monocytes were examined by i.v. transfusion of ex vivo trained monocytes by 4-PBA into recipient high fat diet-fed ApoE -/- mice. Results: In this study, we found that monocytes can be potently reprogrammed by 4-PBA into an immune-resolving state characterized by reduced adhesion and enhanced expression of anti-inflammatory mediator CD24. Mechanistically, 4-PBA reduced the expression of ICAM-1 via reducing peroxisome stress and attenuating SYK-mTOR signaling. Concurrently, 4-PBA enhanced the expression of resolving mediator CD24 through promoting PPARγ neddylation mediated by TOLLIP. 4-PBA trained monocytes can effectively propagate anti-inflammation activity to neighboring monocytes through CD24. Our data further demonstrated that 4-PBA trained monocytes effectively reduce atherosclerosis pathogenesis when administered in vivo . Conclusion: Our study describes a robust and effective approach to generate resolving monocytes, characterizes novel mechanisms for targeted monocyte reprogramming, and offers a precision-therapeutics for atherosclerosis based on delivering reprogrammed resolving monocytes.

Altered DNA methylation underlies monocyte dysregulation and innate exhaustion memory in sepsis.

Innate immune memory is the process by which pathogen exposure elicits cell-intrinsic states to alter the strength of future immune challenges. Such altered memory states drive monocyte dysregulation during sepsis, promoting pathogenic behavior characterized by pro-inflammatory, immunosuppressive gene expression in concert with emergency hematopoiesis. Epigenetic changes, notably in the form of histone modifications, have been shown to underlie innate immune memory, but the contribution of DNA methylation to this process remains poorly understood. Using an ex vivo sepsis model, we discovered broad changes in DNA methylation throughout the genome of exhausted monocytes, including at several genes previously implicated as major drivers of immune dysregulation during sepsis and Covid-19 infection (e.g. Plac8 ). Methylome alterations are driven in part by Wnt signaling inhibition in exhausted monocytes, and can be reversed through treatment with DNA methyltransferase inhibitors, Wnt agonists, or immune training molecules. Importantly, these changes are recapitulated in septic mice following cecal slurry injection, resulting in stable changes at critical immune genes that support the involvement of DNA methylation in acute and long-term monocyte dysregulation during sepsis.

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

DNA methylation reprogramming of genomic imprints in the mammalian germline: A TET-centric view.

The mammalian genome undergoes extensive epigenetic reprogramming twice during development, once during gestation when primordial germ cells (PGCs) are specified from somatic cells and a second time after fertilization in the preimplantation embryo. PGC differentiation into germ cells involves DNA demethylation and subsequent remethylation. DNA demethylation takes place in two waves in the mouse germline, an early phase where most of the genome is demethylated by replication coupled passive demethylation, and a second phase predominated by active DNA demethylation. Imprinted genes, CpG islands on the inactive X chromosome of females, and germline-specific genes are among those loci that are demethylated late. The Ten-Eleven Translocation (TET) family of 5 mC dioxygenases has emerged as active demethylating enzymes that are critical to achieving a DNA hypomethylated state, with TET1 being the most important for imprinted genes. Here, we discuss DNA methylation dynamics in the mammalian genome, with a particular emphasis on DNA demethylation in the germline and the requirement for TET1 in imprinted gene reprogramming.

HNN-core: A Python software for cellular and circuit-level interpretation of human MEG/EEG.

HNN-core is a library for circuit and cellular level interpretation of non-invasive human magneto-/electro-encephalography (MEG/EEG) data. It is based on the Human Neocortical Neurosolver (HNN) software (Neymotin et al., 2020), a modeling tool designed to simulate multiscale neural mechanisms generating current dipoles in a localized patch of neocortex. HNN's foundation is a biophysically detailed neural network representing a canonical neocortical column containing populations of pyramidal and inhibitory neurons together with layer-specific exogenous synaptic drive (Figure 1 left). In addition to simulating network-level interactions, HNN produces the intracellular currents in the long apical dendrites of pyramidal cells across the cortical layers known to be responsible for macroscopic current dipole generation.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

Human Neocortical Neurosolver (HNN), a new software tool for interpreting the cellular and network origin of human MEG/EEG data.

Magneto- and electro-encephalography (MEG/EEG) non-invasively record human brain activity with millisecond resolution providing reliable markers of healthy and disease states. Relating these macroscopic signals to underlying cellular- and circuit-level generators is a limitation that constrains using MEG/EEG to reveal novel principles of information processing or to translate findings into new therapies for neuropathology. To address this problem, we built Human Neocortical Neurosolver (HNN, https://hnn.brown.edu) software. HNN has a graphical user interface designed to help researchers and clinicians interpret the neural origins of MEG/EEG. HNN's core is a neocortical circuit model that accounts for biophysical origins of electrical currents generating MEG/EEG. Data can be directly compared to simulated signals and parameters easily manipulated to develop/test hypotheses on a signal's origin. Tutorials teach users to simulate commonly measured signals, including event related potentials and brain rhythms. HNN's ability to associate signals across scales makes it a unique tool for translational neuroscience research.

Neurons carry information in the form of electrical signals. Each of these signals is too weak to detect on its own. But the combined signals from large groups of neurons can be detected using techniques called EEG and MEG. Sensors on or near the scalp detect changes in the electrical activity of groups of neurons from one millisecond to the next. These recordings can also reveal changes in brain activity due to disease. But how do EEG/MEG signals relate to the activity of neural circuits? While neuroscientists can rarely record electrical activity from inside the human brain, it is much easier to do so in other animals. Computer models can then compare these recordings from animals to the signals in human EEG/MEG to infer how the activity of neural circuits is changing. But building and interpreting these models requires advanced skills in mathematics and programming, which not all researchers possess. Neymotin et al. have therefore developed a user-friendly software platform that can help translate human EEG/MEG recordings into circuit-level activity. Known as the Human Neocortical Neurosolver, or HNN for short, the open-source tool enables users to develop and test hypotheses on the neural origin of EEG/MEG signals. The model simulates the electrical activity of cells in the outer layers of the human brain, the neocortex. By feeding human EEG/MEG data into the model, researchers can predict patterns of circuit-level activity that might have given rise to the EEG/MEG data. The HNN software includes tutorials and example datasets for commonly measured signals, including brain rhythms. It is free to use and can be installed on all major computer platforms or run online. HNN will help researchers and clinicians who wish to identify the neural origins of EEG/MEG signals in the healthy or diseased brain. Likewise, it will be useful to researchers studying brain activity in animals, who want to know how their findings might relate to human EEG/MEG signals. As HNN is suitable for users without training in computational neuroscience, it offers an accessible tool for discoveries in translational neuroscience.

Non-accidental harms ('abuse') in athletes with impairment ('para athletes'): a state-of-the-art review.

OBJECTIVE: Para athletes reap significant health benefits from sport but are vulnerable to non-accidental harms. Little is known about the types and impacts of non-accidental harms Para athletes face. In this literature review, we summarise current knowledge and suggest priorities for future research related to non-accidental harms in Para athletes. DESIGN: Six electronic databases were searched between August and September 2017. 2245 articles were identified in the initial title/abstract review, and 202 records were selected for full-text review following preliminary screening. Two independent examiners evaluated each full text, and eight citations were selected based on inclusion/exclusion criteria. DATA SOURCES: MEDLINE, Embase, PsycInfo, Cumulative Index to Nursing and Allied Health Literature, Scopus and Academic Search Premier. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Inclusion criteria: (A) human participants; (B) written in English; (C) descriptive, cohort and case series, case-control, qualitative, mixed methods studies and all clinical trials; and (D) data pertain to harassment/abuse of youth, recreational, collegiate, national-level and/or elite-level athletes with a physical and/or intellectual impairment. RESULTS: Most studies focused on young, visually impaired athletes and approximately half of all studies described high rates of bullying and its social implications. One study confirmed remarkably high rates of psychological, physical and sexual harms in Para athletes, compared with able-bodied peers. CONCLUSIONS: Bullying in young, visually impaired athletes is described most commonly in the available literature. Due to the limited amount of data, the prevalence of non-accidental harms in Para athletes remains unclear and information on trends over time is similarly unavailable.

Dietary cholesterol and apolipoprotein A-I are trafficked in endosomes and lysosomes in the live zebrafish intestine.

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structural component of high-density lipoproteins. We developed a novel assay to visualize live dietary cholesterol trafficking in the zebrafish intestine by feeding TopFluor-cholesterol (TF-cholesterol), a fluorescent cholesterol analog, in a lipid-rich, chicken egg yolk feed. Quantitative microscopy of transgenic zebrafish expressing fluorescently tagged protein markers of early, recycling, and late endosomes/lysosomes provided the first evidence, to our knowledge, of cholesterol transport in the intestinal endosomal-lysosomal trafficking system. To study APOA-I dynamics, transgenic zebrafish expressing an APOA-I fluorescent fusion protein (APOA-I-mCherry) from tissue-specific promoters were created. These zebrafish demonstrated that APOA-I-mCherry derived from the intestine accumulated in the liver and vice versa. Additionally, intracellular APOA-I-mCherry localized to endosomes and lysosomes in the intestine and liver. Moreover, live imaging demonstrated that APOA-I-mCherry colocalized with dietary TF-cholesterol in enterocytes, and this colocalization increased with feeding time. This study provides a new set of tools for the study of cellular lipid biology and elucidates a key role for endosomal-lysosomal trafficking of intestinal cholesterol and APOA-I. NEW & NOTEWORTHY A fluorescent cholesterol analog was fed to live, translucent larval zebrafish to visualize intracellular cholesterol and apolipoprotein A-I (APOA-I) trafficking. With this model intestinal endosomal-lysosomal cholesterol trafficking was observed for the first time. A new APOA-I fusion protein (APOA-I-mCherry) expressed from tissue-specific promoters was secreted into the circulation and revealed that liver-derived APOA-I-mCherry accumulates in the intestine and vice versa. Intestinal, intracellular APOA-I-mCherry was observed in endosomes and lysosomes and colocalized with dietary cholesterol.

Syndromic surveillance using minimum transfer of identifiable data: the example of the National Bioterrorism Syndromic Surveillance Demonstration Program.

Several health plans and other organizations are collaborating with the Centers for Disease Control and Prevention to develop a syndromic surveillance system with national coverage that includes more than 20 million people. A principal design feature of this system is reliance on daily reporting of counts of individuals with syndromes of interest in specified geographic regions rather than reporting of individual encounter-level information. On request from public health agencies, health plans and telephone triage services provide additional information regarding individuals who are part of apparent clusters of illness. This reporting framework has several advantages, including less sharing of protected health information, less risk that confidential information will be distributed inappropriately, the prospect of better public acceptance, greater acceptance by health plans, and less effort and cost for both health plans and public health agencies. If successful, this system will allow any organization with appropriate data to contribute vital information to public health syndromic surveillance systems while preserving individuals' privacy to the greatest extent possible.