Cellular senescence is recognized as a dynamic process in which cells evolve and adapt in a context dependent manner; consequently, senescent cells can exert both beneficial and deleterious effects on their surroundings. Specifically, senescent mesenchymal stromal cells (MSC) in the bone marrow (BM) have been linked to the generation of a supporting microenvironment that enhances malignant cell survival. However, the study of MSC’s senescence role in leukemia development has been straitened not only by the availability of suitable models that faithfully reflect the structural complexity and biological diversity of the events triggered in the BM, but also by the lack of a universal, standardized method to measure senescence. Despite these constraints, two- and three dimensional in vitro models have been continuously improved in terms of cell culture techniques, support materials and analysis methods; in addition, research on animal models tends to focus on the development of techniques that allow tracking leukemic and senescent cells in the living organism, as well as to modify the available mice strains to generate individuals that mimic human BM characteristics. Here, we present the main advances in leukemic niche modeling, discussing advantages and limitations of the different systems, focusing on the contribution of senescent MSC to leukemia progression. Since Schofield drew up his theory about the existence of a physical hematopoietic niche that drives the hematopoietic stem cell (HSC) fate , the posIn general terms, the available data indicate that alterations in the BM microenvironment can modify the signals received by stroma cells, which, in turn, can induce novel signals that support the expansion of altered hematopoietic cell subsets that eventually become more frequent and damaging ,14,15,16In the BM, mesenchymal stromal cells (MSC) are major and relevant components of the HSC niche, with some of them retaining their colony-forming potential and self-renewal properties; they also have the ability to differentiate into osteogenic, condrogenic and adipogenic lineages. In addition to their proliferative and differentiation potential, MSC are able to modulate neighboring cells behavior through direct contact and paracrine and immunomodulatory signaling ,28,29,30Leukemic stem cells (LSC) share several biological features with HSC, but mainly differ in the impaired regulation of signaling pathways controlling proliferation, survival and invasion ; they arNiche factors, such as osteopontin, CXCL12 and cytokines secreted by MSC, osteoblasts and endothelial cells may induce migration of LSC, activate signaling pathways, promote cell proliferation, and inhibit apoptosis. Niche factors can also activate LSC intrinsic self-renewal pathways, enhancing LSC survival as it occurs in AML  or prediInterestingly, healthy MSC co-cultured with B cell acute lymphoblastic leukemia (B-ALL) cells show senescent hallmarks that are similar to cultured MSC isolated from leukemic patients . MoreoveSenescence is induced by several types of stress  that trigger the activation of p16INK4A/Rb or p53/p21CIP1 signaling pathways. Phenotypically, it is characterized by changes in chromatin organization and gene expression that dramatically modify metabolic and secretory features in cells , resultiIn order to study these phenomena, and because of the dual role of the niche in regulating normal and malignant hematopoiesis, a rigorous assessment of the BM cell population diversity and the molecular mechanisms of MSC senescence are required to enable assays where alterations in niche cells can be experimentally tuned, and the consequences on HSC maintenance and leukemic stem cell (LSC) development can be determined. However, the understanding about cells and factors controlling human normal and malignant hematopoiesis in the BM remains limited, due the difficulty of modeling cellular relationships in the human system. Because of this, the knowledge about HSC biology and the contribution of altered MSC to cancer progression is mainly based on in vitro and in vivo murine models that have been extensively used in order to examine the physiology and relationships among BM niche cells and their role on HSC and LSC expansion . In thisIn this review, we present recent advances in leukemic niche modeling, discussing advantages and limitations of in vivo and in vitro systems. We focus on the contribution of senescent MSC to leukemia progression in these models. Understanding the role of senescent MSC in leukemia development may be useful to identify new therapeutic strategies to limit malignant cell survival and reduce treatment resistance, even by remodeling the surroundings toward a pro-normal microenvironment that is able to neutralize the leukemic niche influence and to restore HSC functioning.The design of accurate in vitro models is a compulsory tool for basic research; the in vitro studies enable comparisons among different cell types, donors or donor´s ages; they also have the advantage of allowing direct cell manipulation, controlling most experimental variables, studying isolated responses and facilitating quantitative analysis. These models for cancer research vary in complexity, ranging from tumor-derived cell lines to reconstruction of 3D models of the tumor microenvironment .The better understanding of both HSC and niche components biology has allowed for significant advances regarding the successful HSC expansion ex vivo, in spite of the remaining questions about the mechanisms of HSC regulation in vivo. Conventional HSC culture systems are expanded on polystyrene plates in optimized culture media, whereas the long-term maintenance of LSC is still difficult, due to the difficulty of preventing blast apoptosis to stop spontaneous differentiation and to minimize the effect of inhibitory feedback signals by differentiated cells in the culture. Patient-derived and immortalized leukemic cell lines are frequently used in experimental assays; however, their characteristics do not correspond to a functionally major LSC population ,35,68.The addition of support cells  and the variation in biophysical parameters on the substrate to conventional 2D cultures have enabled the culture and maintenance of leukemic cells; in spite of their simplicity, noteworthy advances have been achieved by using screening approaches and culture systems supplemented with cytokines and support cells under proper oxygen and metabolic conditions. Major advances in understanding leukemic cell biology, cancer development and chemoresistance have been carried out, recreating the leukemic microenvironment in 2D culture systems for B-ALL ,70,71,72The use of support cells in 2D systems is still the primary approach used to maintain HSC and LSC cultures. Several studies have shown the importance of MSC as niche supportive cells and providers of regulator factors for human HSC and LSC ,87, as wMSC are rare within the BM aspirate; therefore, they must be expanded in vitro before they can be used for research or therapeutic purposes and it is clear that culture conditions and metabolic cell state can critically affect their role in supporting leukemic cells. For example, it has been shown that tension or elasticity changes in the culture substrate can rule MSC differentiation fate ,92. It wAdditionally, most of the protocols add fetal bovine serum (FBS) as a media supplement for MSC, but its use has some disadvantages, such as the induction of side immunological effects, possible microbial exposure and batch variability. A study with the aim of evaluating the differences on the onset of replicative senescence between MSC cultured with FBS or human autologous serum showed that the serum source was a determinant for the time until reaching senescence and the proportion of cells in S-phase during early passages .Senescence-related features in stroma cells appear to be frequently associated with conditions favoring LSC expansion in BM niche models, and several studies have addressed the question of the influence of an aged or senescent microenvironment on disease progression ,97,98,99In spite of their great utility, 2D culture systems show some constraints that limit their use to accurately reflect in vivo conditions such as the reduced availability of oxygen, the non-continuous nutrients’ access, the mechanical forces exerted by the liquid components , the spaHence, several protocols for bioengineering 3D HSC niches have been established for mouse and human HSC and LSC expansion ,110,111.Inorganic , natural  or synthetic polymers  have been tested as scaffolds to provide a 3D structure for normal and leukemic niches (reviewed by ); polymeImprovements to 3D culture systems were developed in order to capture and perform single-cell analysis on HSC cells maintained in microfluidic devices , or to cMuch of the research regarding leukemic niche modeling is related to the screening of therapeutic compound targeting of LSC in the BM . Using aIn spite of the advances on 2D and 3D in vitro models, they still have physiological constraints because they failed to capture the whole complexity of the tumor microenvironment; their biggest limitation relies on the lack of correspondence with a whole organism. This limits the use of the in vitro models for some experimental approaches , making in vivo studies in animal models necessary.Macaca mulatta) have been used, but on a considerably lesser scale than mice and rats  pathway plays a key role on vertebrate senescence was previously discovered in yeast, nematodes and flies ; howeveriewed by ,127,128)Rattus norvegicus, mainly Fischer 344, Sprague-Dawley, Wistar, and Long-Evans strains), Syrian hamster (Mesocricetus auratus), the naked mole rat , and the Mongolian gerbil (Meriones unguicultatus), have been used as models for aging and senescence studies . Besides several mice strains that are discussed in more detail below, other small rodents, such as rats .Leukemic mice models are generated by exposure to carcinogenic chemicals, genomic integration of oncogenes delivered by virus or transposons, implantation of modified embryos and zygotes transplanted with ex vivo manipulated HSC (transgenic models) or injection of primary patient samples or human cell lines into immunocompromised host animals (xenograft models)  (reviewenullIL2rγ) and the combination with mutations in the protein kinase DNA-activated catalytic polypeptide gene (scidPrkdc or scid) or in the recombination activating gene 1 or 2  that allowed the generation of mice lacking adaptative immunity and exhibiting deficiencies of innate immunity. Immunodeficient nullIL2rγ mice have enabled the successful engraftment of various human cancers; the strains NSG (NOD.scidIl2rγtm1WjlCgPrkdc), NOD (NOD.scidIl2rγtm1SugCg-Prkdc) and BRG  are the three kinds of immunodeficient nullIL2rγ mice more frequently used as in vivo platforms for identifying and testing drug targets, for the identification of LSC and investigation of metastasis, as well as preclinical models for the evaluation of new therapeutics [Xenograft mice models arose after the development of immunodeficient mice bearing the IL-2 receptor common gamma chain  when co-cultured with B-ALL cells and that periostin deficiency in MSC decreases CCL2 expression in co-cultured B-ALL cells; CCL2 expression in these cells is regulated by the ILK/NF-kB pathway . In a SCNras-mutant mouse model, it was experimentally proved that a senescent BM microenvironment promotes a chronic myelomonocytic leukemia-like condition and that intra-BM transfusion of young MSCs can rejuvenate the senescent BM microenvironment [Second, many of the physiological changes described for MSC in pro-leukemic and leukemic microenvironments are equivalent to those occurring during BM-aging and senescence  and it hironment . Howeverironment .INK4a expression in stromal cells within the BM, but also that depletion of SASP and senescent stromal cells promote leukemic mice survival [INK4a in a cohort of mice showed strong induction of p16INK4a in the healthy stroma of early neoplasms, but a lack of association between the expression of this senescence marker and cancer-related mortality [INK4A expression can be induced in non-senescent macrophages as part of a reversible polarization response to immune stimuli in mice, with hemizygous p16INK4a knock-in of the luciferase gene reporter [INK4a, other genes associated to senescence in mice do not have similar expression patterns in humans, as it happens with the alternative products of INK4 locus (p16 and ARF) [To better investigate how stromal dysfunction can foster the development of cancer and to elucidate the cellular and molecular mechanisms of MSC senescence that contribute to leukemia progression, other approaches relying on senescent cell identification and ex vivo characterization using fluorescent tags , as wellsurvival . Howeverortality . Here, ireporter . In addiand ARF)  and Rb gand ARF) .INK4a up regulation, cell cycle arrest after chemotherapy and decreased regenerative capacity) and activation of p38MAPK/FOXO1, a ROS concentration-dependent signaling pathway [+ as antioxidant protection for leukemic cells against chemotherapy [Reactive oxygen species (ROS) signaling clues are important to maintain quiescence and define differentiation fates of HSC and LSC , and als pathway . A combi pathway . Also footherapy . In a stotherapy .The relevance of mice models for studying telomere-related human biology is also debated ,189,190.−/− individuals from the F1 and F3 generation (with differences in telomere length and telomerase activity) and the progeny of different back-crosses between them, the researchers concluded that leukemia can be induced in the absence of telomerase activity and the aging microenvironment induced by telomere dysfunction can accelerate it; however, short telomeres can prevent leukemogenesis by inducing DNA damage [The use of mice models of oncogenesis led to the theory that telomerase deficiency operates initially as a pro-oncogenic pathway, probably through chromosomal instability but continuous inactivation of telomerase in tumor cells would decrease tumor growth . So, micA damage . ConversA damage . A previA damage . TelomerA damage ).2 availability in murine BM and found a greater frequency of hypoxic regions in advanced-stage B-ALL individuals [Another interesting approach to study biological processes in vivo, including hematopoietic, senescence and leukemic pathways, is the use of two-photon microscopy ,201,202.ividuals .As already described, traditional and cutting-edge technologies used for senescence assessment and BM modeling support the key role of senescent/aging BM microenvironment-related alterations in the initiation and progression of hematological malignancies. Future research could be used, not only to improve the understanding of HSC regulation by the niche or to better elucidate the molecular mechanisms that swing cells targeted by senescence clues toward apoptotic or malignant transformation, but also to modulate or to revert senescence progress at different stages, to evaluate how the SASP impacts the interaction between senescent cells and the immune system over time, and to improve current therapies against leukemias using senolytic approaches. In fact, preclinical models suggest that senolytic drugs would be able to decrease the in vivo frequency of senescent cells, reduce inflammation and improve patients welfare (reviewed by ). In thiIn recent years, there has been a growing interest regarding the role of the senescent BM microenvironment in leukemia development, but the causal relationship between both events is still unclear, partly due to the wide range of in vitro and in vivo models available for leukemia study and the variety of protocols used for senescence study. In spite of their value, as tools for basic research and pre-clinical trials for drug efficacy evaluation, there is still a need for better BM leukemic niche modeling, as well as a standardized methodology for cellular senescence assessment. In the meantime, and because different methods of inducing senescence have singular characteristics and effects, care must be taken to ensure that the chosen model is well-suited to the experiment and its objective. Specifically, it is a matter of discussion regarding how representative mouse models of hematopoietic aging or leukemia development are and if further modeling should be conducted in aged mice instead of young animals to understand the context dependency of genetic and pharmacological perturbations. This is particularly relevant when discussing murine models for pediatric leukemias, where differential effects of young vs. old mice’s BM microenvironment on B-ALL cells can be observed . Concern


Despite aluminum’s prevalence and abundance in the biosphere it is remarkably well-tolerated by all plant and animal species; no organism is known to utilize aluminum metabolically; however, a biological role for aluminum has been assigned in the compaction of chromatin. In this Communication, several examples are given where aluminum has been shown to irreversibly perturb and/or stabilize the natural conformation of biomolecules known to be important in energy metabolism, gene expression, cellular homeostasis and pathological signaling in neurological disease. Several neurodegenerative disorders that include the tauopathies, Alzheimer’s disease and multiple prion disorders involve the altered conformation of naturally occurring cellular proteins. Based on the data currently available we speculate that one way aluminum contributes to neurological disease is to induce the misfolding of naturally occurring proteins into altered pathological configurations that contribute to the neurodegenerative disease process.The natural element aluminum possesses a number of unique biochemical and biophysical properties that make this highly neurotoxic species deleterious towards the structural integrity, conformation, reactivity and stability of several important biomolecules. These include aluminum’s  In fact aluminum, as alum  is used globally to aggregate organic/biological impurities in turbid drinking water in order to precipitate them and to clarify an unappealing cloudy water product into a clear, finished water product  and computer-aided algorithms, statistical and imaging analysis, preliminary unpublished evidence from our laboratory suggests that aluminum 3) at nanomolar concentrations strongly promotes a shift in conformation from the PrPc into the PrPsc isoform both in vitro and in human neuronal-glial (HNG) cells in primary co-culture. As such aluminum salts may be an integral component in the molecular-metallic mechanism involved in inducing protein misfolding disease  of aluminum’s unusually high positive charge density, unique biochemical and biophysical properties under physiological conditions and within complex biological systems; (ii) this metallotoxin’s ubiquity as the most abundant metal in the earth’s biosphere and enrichment in naturally-occurring biological systems; (iii) of aluminum’s wide spread bioavailability in drinking water, food, medicines, cosmetics and other consumer products and in atmospheric dust and groundwater that promotes extensive human exposure; (iv) multiple investigators have demonstrated aluminum’s extreme neurotoxicity on multiple neurobiological systems, even at very low ambient (nM or lower) levels; and (v) of aluminum’s ability to aggregate naturally occurring cellular proteins and peptides, a process known to drive neuro-inflammation, alter neurogenesis and neuronal cytoarchitecture and induce significant memory and learning deficits in multiple experimental murine models [There has long been an interest in aluminum as a pathogenic factor in human biology, and especially in neurobiology and neurology, largely because: e models ,59,60,61Although aluminum is especially abundant in the biosphere and is remarkably well tolerated by all plant and animal species, no organism is known to use aluminum in any active metabolic process. There is, however, abundant evidence for a biological role for aluminum in the packaging of genes into inactive chromatin conformation ,16,17,32(i) the interaction of aluminum with other environmentally-available metals such as mercury, which together exhibit significantly synergistic and neurotoxic effects [(ii) aluminum’s effects on nucleoprotein modifications including the epigenetic modification of histones and other DNA-associated nucleoproteins [(iii) the association and/or interaction of aluminum-modified and/or misfolded proteins with the pathological hallmarks of neurodegenerative disease that include prions, Aβ peptides, tau proteins and α-synuclein [(iv) the neutralization of aluminum neurotoxicity and genotoxicity including multiple chelation approaches and the use of both synthetic and naturally occurring compounds [(v) the potential for the interaction of aluminum with other oxygen-donor ligands which occur at relatively high densities within the natural cellular and nuclear compartments of all eukaryotic organisms.In conclusion, the pathological role for aluminum in neurological disorders has been controversial largely due to an incomplete understanding of aluminum’s precise mode of interaction with select biomolecules and its targeting to specific biological compartments. Many independent researchers have uncovered highly interactive aspects of aluminum neurotoxicity and the precise details of these molecular-metallic pathological mechanisms are becoming increasingly clear. Several seriously understudied areas of aluminum neurotoxicology are:  effects ,63; (ii)proteins ; (iii) tynuclein ,37,38,65ompounds ,67,68,69


Mitochondria are ATP-generating organelles in eukaryotic cells that produce reactive oxygen species (ROS) during oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA) is packaged within nucleoids and, due to its close proximity to ROS production, endures oxidative base damage. This damage can be repaired by base excision repair (BER) within the mitochondria, or it can be degraded via exonucleases or mitophagy. Persistent mtDNA damage may drive the production of dysfunctional OXPHOS components that generate increased ROS, or OXPHOS components may be directly damaged by ROS, which then can cause more mtDNA damage and create a vicious cycle of ROS production and mitochondrial dysfunction. If mtDNA damage is left unrepaired, mtDNA mutations including deletions can result. The accumulation of mtDNA mutations has been associated with conditions ranging from the aging process to cancer and neurodegenerative conditions, but the sequence of events leading to mtDNA mutations and deletions is yet unknown. Researchers have utilized many systems and agents for generating ROS in mitochondria to observe the downstream effects on mtDNA, ROS, and mitochondrial function; yet, there are various drawbacks to these methodologies that limit their precision. Here, we describe a novel chemoptogenetic approach to target oxidative damage to mitochondria and mtDNA with a high spatial and temporal resolution so that the downstream effects of ROS-induced damage can be measured with a high precision in order to better understand the mechanism of mitochondrial dysfunction in aging, cancer, and neurodegenerative diseases. Oxygen is the final electron accepter in the electron transport chain, and the addition of four electrons to the oxygen molecule creates metabolic water. The leakage of electrons from the ETC can generate superoxide radical anions, which can spontaneously or through the action of mitochondrial superoxide dismutase create hydrogen peroxide (H2O2). Superoxide can displace iron from iron sulfur centers in the ETC, inactivating key steps of OXPHOS. H2O2, if not broken down by glutathione peroxidase or peroxiredoxin, can interact with metal cations such as Fe2+ through Fenton chemistry, generating highly reactive carbonate and hydroxyl radicals [Mitochondria are organelles in eukaryotic cells that generate the majority of ATP for the cell through a process called oxidative phosphorylation (OXPHOS). They also play important roles in programmed cell death, the biosynthesis of important metabolites, and the homeostasis of cellular iron and calcium. Mitochondria contain two membranes, and the inner of these two membranes has embedded within in it key protein complexes for OXPHOS. As glucose and other molecules, such as fatty acids, are catabolized by the cell, electrons from these molecules are carried by nicotinamide adenine dinucleotide (NAD) or flavine adenine dinucleotide (FAD) to the OXPHOS machinery on the inner mitochondrial membrane. NAD and FAD then deliver these electrons to complex I (NADH dehydrogenase) and complex II (succinate dehydrogenase), respectively, which are the first two complexes of the five complex electron transport chain (ETC). As electrons are passed to complexes I, III, and IV, protons are simultaneously pumped into the intermembrane space to form an electrochemical gradient. Complex V (ATP synthase) then utilizes this electrochemical gradient, spinning its nanoturbine to generate ATP from ADP + Pradicals . These rEukaryotic cells may contain up to a thousand mitochondria, and each mitochondrion possesses ten to fifteen copies of its own genome. The human mitochondrial genome is only ~16.5 kb in size, as opposed to the 3.3 billion base pairs of nuclear DNA, and encodes for 13 subunits of the OXPHOS machinery as well as two ribosomal RNAs and 22 transfer RNAs. The vast majority of the ~1500 enzymes and protein subunits that function in mitochondria are imported from the cytoplasm and mtDNA is replicated, transcribed, and translated within the organelle.The mitochondrial genome is packaged into nucleoids  and due Ogg1 were found to have accumulated 8-oxoG lesions in their mtDNA [Sod2 gene encoding for the mitochondrial antioxidant enzyme MnSOD display OXPHOS dysfunction, increased glycolysis and decreased ATP levels in their skin cells [Mouse models that seek to elucidate the mechanism of ROS-induced mitochondrial dysfunction have been created. Mice deficient in the BER glycosylase ir mtDNA  and yet,in cells . This in2O2 preferentially damages mtDNA over nDNA. H2O2 is uncharged and can diffuse freely within the cell, damaging components throughout the cell. Using an innovative QPCR assay to follow damage and repair in mtDNA we have shown that H2O2 induces three to 10-fold more damage to mtDNA than nDNA and protracted treatments suggested that the damage was unable to be repaired [2O2 in cells showed similar results, in irreparable mtDNA damage and a temporary decrease in mitochondrial function as evidenced by a decreased in the reduction in MTT [2O2 treatment, the mitochondria exhibiting low mitochondrial membrane potential are those that also exhibit persistent mtDNA damage [2O2 for 60 min leads to persistent mtDNA damage, loss of mtDNA copy number, and dysfunctional OXPHOS, implicating both damage and degradation of mtDNA in loss of mitochondrial function [2O2 suggest that mitochondria unable to repair oxidative mtDNA damage end up with OXPHOS dysfunction and organelle dysfunction in general, and yet similar levels of MMS-induced alkylation damage to mtDNA were able to be repaired efficiently. A major issue with using H2O2 to treat cells include the ability of the H2O2 to diffuse freely throughout the cell and potentially damage many targets, not just mtDNA [To elucidate the relationship of mtDNA damage and OXPHOS deficiency to disease states, several agents and systems have been employed in the past. Treating cells with Hn in MTT . A 2003 A damage . Treatinfunction . Studiesst mtDNA . + acts as a potent complex I inhibitor that results in the production of ROS and impairment of mitochondrial respiration [Other agents have been applied to cells in order to generate mitochondrial ROS, including the complex I inhibitor rotenone and the neurotoxin MPTP . When rotenone was applied to the growth medium of normal or DNA-repair deficient fibroblasts, increased intracellular ROS resulted, as evidenced by the oxidation of dihydroethidium to ethidium, but there was no detectable nDNA damage . While tpiration . In a sepiration . Interespiration .Base damage can also cause replication blocking lesions, which may be overcome by DNA polymerase ϴ translesion synthesis, which might be a source of mtDNA mutations . ReplicaMtDNA damage and dysfunction have been implicated in aging, cancer, and neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Mitochondrial dysfunction in many of these conditions may take decades to appear, which is difficult to reproduce in a lab. Rather, researchers have used many tools and damaging agents in an attempt to replicate the initial insult responsible for the pathogenesis of these conditions, and yet each of these methods has benefits and drawbacks. Data suggest that persistent oxidative damage to mtDNA leads to a downstream dysfunction in OXPHOS leading to a vicious cycle of ROS production and mtDNA damage. This review discusses the various historical methods of replicating mitochondrial dysfunction in the laboratory and introduces a newly developed chemoptogenetic approach to specifically target mtDNA and thus better understand the role of oxidative mtDNA damage in these conditions.Mitochondria-targeting drug conjugates have also been developed to selectively damage this organelle. These include mitochondria-penetrating peptides (MPPs), which accumulate inside mammalian mitochondria and can be conjugated with DNA-damaging agents such as chlorambucil and doxorubicin ,21,22,23C. elegans [C. elegans and may result in cell death in the absence of miniSOG activity [Photosensitizing agents have been used to induce targeted damage to mitochondria and has been the basis of photodynamic therapy . When ph elegans . MiniSOG elegans . In addiactivity . That miactivity .To overcome these problems and to understand the spatiotemporal events leading to mitochondrial dysfunction, we designed a precise, highly controlled chemoptogenetic tool to generate mitochondrial damage that is based on the FAP (Fluorogen Activating Protein) approach, as developed by Professor Marcel Bruchez  Figure . The basOur group has used the mito-FAP system to target singlet oxygen damage to mitochondria . The conAt four hours post-treatment, there was a reduction in oxygen consumption and a concomitant increase in glycolysis indicating mitochondrial OXPHOS dysfunction. No change in mitochondrial bioenergetics was seen in cells that did not express Mito-FAP, indicating that the Mito-FAP construct, in combination with MG2I dye and light, was responsible for the change in bioenergetics. The presence of the singlet oxygen quencher sodium azide during light activation or the ROS scavenger N-acetylcysteine (NAC) during and after light activation was able to partially or completely protect mitochondrial respiration, respectively, indicating that ROS production was causative for mitochondrial dysfunction. Additionally, there was a progressive decline in the activity of several mitochondrial ETC complexes, including complexes I, III, and IV beginning at four hours post-treatment. Western blot indicated a decrease in Complex IV subunit II and Complex I subunit NDUFS3, both nuclear-encoded ETC genes  Figure .To determine whether ROS generation in the mitochondria leads to a change in mitochondrial membrane potential, the cationic fluorescent probe tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used 24 h after the dye and light treatment. A red-to-green shift of JC-1 fluorescence is indicative of a loss of mitochondrial membrane potential, and nearly a third of the Mito-FAP cells with dye and light treatment displayed this shift. In addition to a loss of mitochondrial membrane potential, fragmented mitochondria were observed four hours after treatment, and levels of the phosphorylated mitochondrial fission protein Drp1 increased at 24 h following treatment, indicating increased activity of this protein. Mitochondrial fission has been proposed as a way for the cell to separate any remaining functional mitochondria from irreversibly damaged ones that are destined for mitophagy. In addition to the effects on OXPHOS, mitochondrial dynamics and membrane potential, and ETC complex activity and expression, the dye and light treatment resulted in mtDNA damage that persisted for at least 24 h after the treatment, in contrast to a lack of nDNA damage .2O2 by MnSOD within the mitochondria, we next explored the nature and severity of any extramitochondrial damage resulting from this secondary wave of ROS, including effects on cell growth, nuclear proteins, and telomeres.Protection of loss of mitochondrial function after light and dye treatment of Mito-FAP expressing cells by N-acetylcysteine suggested that ROS-damaged mitochondria generate another wave of ROS that amplifies damage to the mitochondria and to the cell. MitoSOX dye indicated superoxide production in nearly 80% of cells at four hours post-treatment, and this increased superoxide signal persisted for at least 48 h post-treatment. As superoxide is quickly converted to the freely diffusible H2O2, then it might be expected that nuclear proteins and DNA could be damaged. To determine the downstream effects of mitochondrial ROS on the nucleus, we used a ratiometic approach to detect oxidized versus free thiols and determined that nuclear proteins were oxidized by H2O2, in combination with an increase in the pHyper-nuc signal, a fluorescent nuclear H2O2 sensor. Surprisingly, we could not detect gross nuclear DNA damage using the alkali comet assay. However, we found that the dysfunctional mitochondrially generated H2O2 causes a large increase in telomere damage. This may be due to the fact that telomeric sequences readily bind iron and thus are much more susceptible to Fenton chemistry and subsequent hydroxyl radical formation [2O2 preferentially damages telomeres [Cells treated with dye and light showed a significant reduction in cell growth that was not due to apoptosis but instead was caused by cell cycle delay driven by DNA replication stress. If dysfunctional mitochondria generate significant fluxes of Hormation . Forty-eelomeres   in mitochondria that leads to vicious cycles of ROS production and mitochondrial dysfunction in aging and disease processes remains unsolved. One aspect of the Mito-FAP fusion construct is that while a cytochrome oxidase leader sequence was used, the precise mitochondrial matrix location was not known.+/K+ ATPase and neuronal depolarization. A Seahorse flux analyzer was used to measure mitochondrial respiration in paralyzed whole larvae exposed to near-infrared light, and a 24% decrease in oxygen consumption rate was observed in the NeuMito-FAP-MG2I larvae compared to controls. Next, the brain cells of the NeuMito-FAP-MG2I larvae were evaluated in the flux analyzer during and after light treatment, and they showed a dramatic decrease in maximal respiration compared to controls. The loss of basal oxygen consumption and maximal respiration shows that the neuronal mitochondria of the NeuMito-FAP-MG2I were disrupted by the treatment [The mito-FAP system has been extended to ablate mitochondrial function in living zebrafish neurons by Dr. Ed Burton’s laboratory . Double reatment . This stIn order to better understand the precise role that mtDNA damage plays in mitochondrial dysfunction in cancer, neurodegeneration, and aging, it is imperative that new methods are developed to specifically target mtDNA and not nuclear DNA or other macromolecule components . MitochoPreliminary studies in our lab show that a single treatment with dye and 660 nm light reduces the growth of our HEK293 TFAM-FAP cells, as measured by CyQuant. We have also observed mitochondrial dysfunction after multiple treatments with light plus dye, specifically, uncoupled mitochondrial function as measured in the Seahorse extracellular flux analyzer. Future studies involve quantifying the level of 8-oxoG lesions in mtDNA with quantitative long-amplicon PCR  followinAn accumulation of mtDNA deletions has been observed in older people, Parkinson’s disease (PD) patients, and Alzheimer’s disease (AD) patients . The submt). The UPRmt has several downstream mediators, including mitochondrial sirtuin SIRT3, which activates FOXO3a, followed by the activation of antioxidants including manganese superoxide dismutase (SOD2) and the removal of badly damaged mitochondria via mitophagy [mt has been suggested to lead to mitochondrial biogenesis and replication that is not selective for particular mtDNA molecules, allowing for wild-type and mutant mtDNA genomes to be replicated and for the vicious cycle to continue [mt are yet unknown [Mutations in mtDNA have also been linked to carcinogenesis and are found in a wide variety of cancers ,46. Inteitophagy . The indcontinue . Even so unknown , and so The concept that mtDNA mutations are driven primarily by oxidative damage has been challenged more recently. A 2020 study by Yuan et al. performed the whole-genome sequencing of mtDNA from malignant tumors and from aging tissues and proposed that it is, in fact, errors in mtDNA replication, and not oxidative lesions, that are the primary cause of mtDNA mutations in malignant tumors. This was namely due to their finding mutations that were predominantly strand-asymmetric substitutions of C to T and of T to C, rather than the major ROS-induced mutation of G to T in these tumor tissues, as driven by the 8-oxoG lesion . Recent Studies that detect mtDNA deletions and/or mutations in diseased tissues of patients are unable to elucidate the mechanism of the damage leading to these disease states, but rather only provide information about the outcome. Thus, these retrospective studies, while informative, lack clear cause and effect relationships, and three key questions need to be explored: (1) do dysfunctional and/or damaged OXPHOS components generate ROS that then preferentially target mtDNA, (2) does damaged mtDNA lead to damaged OXPHOS components that then generate a vicious cycle of increased ROS and mtDNA damage, and (3) what is the nature and severity of mtDNA damage that leads mitochondria to degrade their DNA rather than repair it?To directly address these questions, we require a system that can specifically and precisely oxidize mtDNA and not nuclear DNA. It is also imperative to understand what particular aspects of mtDNA damage induce the degradation versus the repair of mtDNA lesions in order to elucidate how this decision impacts the pathogenesis of neurodegenerative diseases and cancers.The singlet oxygen generated by the chemoptogenetic system produces almost exclusively 8-oxoG damage in DNA. By coupling the chemoptogenetic system to TFAM, an abundant protein tightly associated with mtDNA, we can generate singlet oxygen in close proximity to mtDNA and generate the 8-oxoG lesion at high levels in the mitochondrial genome. High levels of 8-oxoG in mtDNA have been associated with mtDNA mutation, deletion, apoptosis, mitochondrial fragmentation, and mitochondrial dysfunction ,61, and


Cutaneous metastases have distinct morphologic features that can aid in making the diagnosis clinically even prior to biopsy. Lesions often have a nodular appearance and are firm, fixed, and range from flesh-colored to reddish-purple. A 73-year-old female with a history of lung adenocarcinoma status-post neoadjuvant chemotherapy and lobectomy 20 months prior was referred to our dermatology clinic for evaluation and treatment of suspected keloids on the left flank. The lesions were firm, plum-colored, fibrotic nodules, and were diagnosed clinically in the office as cutaneous metastases of internal malignancy. Punch biopsy was performed and revealed a proliferation of atypical epithelial cells arranged in cords and strands, with neoplastic cells positive for CK7 and TTF-1, confirming the diagnosis of metastatic adenocarcinoma. The patient was referred for chemotherapy and is still alive nine months following the prompt clinical diagnosis of cutaneous metastasis. Cutaneous metastasis signifies a poor prognosis, but knowledge of the clinical characteristics of these lesions can lead to earlier detection and more prompt initiation of treatment. The skin is an uncommon site of metastasis from internal malignancies, with cutaneous metastases encountered in 1-10% of all patients with cancer . in the setting of lung cancer, the most frequent sites of metastasis are the brain, bone, liver, and adrenal glands. Skin metastases occur much less frequently, with one meta-analysis showing an incidence of 3.4% in patients with lung cancer. Cutaneous metastases in the setting of lung cancer most commonly present as firm, painless, nodules on the chest, abdomen, or head and neck region, but can present in a variety of forms and mimic benign lesions . CutaneoWe present a case of a 73-year-old female with a history of adenocarcinoma of the lung who was initially referred for evaluation and treatment of keloids but was diagnosed with metastatic adenocarcinoma. The aim of this case is to promote awareness of the morphology of skin metastasis so that physicians can diagnose these lesions clinically with minimal delay in diagnosis and treatment.A 73-year-old female with a history of lung adenocarcinoma status-post neoadjuvant chemotherapy (three cycles of cisplatin/nivolumab/pemetrexed) and left upper lobectomy 20 months prior with adjuvant radiation was referred by her primary care physician for evaluation of suspected keloids on the left side of her abdomen Figures -1C. The A 4-mm punch biopsy of the lesion was performed given the suspicion for malignancy. Biopsy results revealed ulceration of the surface with an inflammatory crust, along with a proliferation of atypical epithelial cells present diffusely between and among collagen bundles of the dermis. The neoplastic cells were arranged in cords and strands and positive for CK7 and TTF-1, and negative for CK20. This pattern is suggestive of metastatic adenocarcinoma and was diagnosed as so in the pathologic report, compatible with the primary lung cancer. Two days following the presentation to our clinic, the patient presented to an academic medical center with worsening shortness of breath. CT scan revealed a large loculated left pleural effusion, significantly increased in size compared to imaging seven months prior Figures -2B. A viThe skin is an uncommon site of metastasis when compared with other organs. Mollet et al. showed in a systematic review of cutaneous metastasis that the most common sources of cutaneous metastases in men are the lung (24%), colon (19%), melanoma (13%), and oral cavity (12%). The most frequent malignancies that metastasize to the skin in women are breast 69%), colon (9%), melanoma (5%), ovarian (4%), and lung cancer (4%) [9%, colonThe appearance of cutaneous metastases varies widely and can mimic benign skin lesions, particularly when they are a single lesion . The mosIn our patient, the lesions were initially diagnosed as keloids prior to referral to our clinic. Although keloids can also present as firm, pink, nodules and be similar in size to cutaneous metastases, keloids are softer and directly overlay surgical scars, skin piercings, or other areas of skin trauma. The lesions present on our patient were adjacent to her prior surgical scar, rather than directly overlying it. One study showed that 86% of patients with keloids complained of pruritus, while 46% experienced pain . KeloidsThe anatomical distribution of the cutaneous metastasis often correlates with the origin of primary malignancy and the mechanism of metastatic spread. Internal malignancies generally metastasize to sites close to the primary tumor but are capable of metastasizing anywhere on the cutaneous surface. Lung, melanoma, and breast cancers are the most common malignancies to metastasize to remote cutaneous sites . The mosImmunohistochemical markers can assist in the identification of the primary cancer, as cutaneous metastases from internal malignancies are indistinguishable from one another by their appearance on a physical exam alone. Expression of CK7 is positive in cancers of the lung, breast, ovary, pancreas, thyroid, and salivary gland, whereas expression of TTF-1 is characteristic of primary lung cancer and thyroid cancer. When a thyroid primary is ruled out, TTF-1 expression is both sensitive and specific for primary adenocarcinomas, bronchioloalveolar carcinomas, and small-cell carcinomas . ExpressCutaneous metastases are a sign of poor prognosis. A retrospective analysis of patients with biopsy-proven skin metastases performed by Sariya et al. showed that 86% (43/50) of patients with cutaneous metastases had known stage IV cancer, and the mean interval between diagnosis of the primary malignancy and the development of skin metastasis was 36 months (ranging from 1-177 months) -15. TherCutaneous metastases from the lung are relatively uncommon and are a sign of poor prognosis. They most commonly present as nodular lesions on the chest, abdomen, or head and neck region, but can present in a variety of other forms and on any cutaneous surface. Although metastases can mimic benign lesions such as keloids, there are distinct clinical differences that can help clinicians to diagnose metastases early and limit delays in initiation of treatment. Lesions that are painless, firm, fixed, or associated with secondary ulceration, especially in combination with a history of internal malignancy should raise suspicion for cutaneous metastasis. In patients with a history of smoking or lung cancer, any suspicious skin lesions should prompt full thickness skin biopsy using a punch tool or incisional biopsy for histological diagnosis of possible metastasis.


Additional concern on this matter comes from the preclinical and clinical evidence showing that several of the drugs that are used for recreational purposes may have central and peripheral toxic effects of variable severity, some of which may persist even after drug discontinuation . The picSmith et al. investigated the effects of smoked cannabis on epigenetic systems, immunological parameters and cannabinoid receptors (CBR) of type 1 and 2 in human peripheral blood cells of regular cannabis users. They found that mRNA for CBR2 increased in blood lymphocytes of cannabis users who had a higher blood concentration of Δ9-tetrahydrocannabinol (THC), compared with cannabis users who had a lower THC blood concentration. However, such an increase in the mRNA for CBR2 was not accompanied by changes in immunological markers. Moreover, changes in mRNA for methylation and demethylating enzymes  were observed in lymphocytes of cannabis users, suggesting that THC may elicit epigenetic changes. Since increases in DNMT have been linked to some of the pathophysiological processes featuring schizophrenia, the authors suggest that the effects of cannabis observed here should be further investigated in a larger cohort, to ascertain if changes in the levels of DNMT enzymes may be one of the potential mechanisms by means of which cannabis can trigger schizophrenia in vulnerable individuals.Kazmi et al. performed an exploratory analysis to investigate the existence of associations between pro-inflammatory cytokine markers and subjective measures of sleep quality, scores of anxiety/depression and alcohol consumption among individuals with alcohol use disorder. The results obtained in this study demonstrate that the levels of the cytokines Monocyte Chemoattractant Protein-1 (MCP-1) and interleukin-8 (IL-8) may be associated with higher drinking behavior, poorer sleep quality, and higher anxiety/depression in individuals with alcohol use disorder. Moreover, markers of inflammation and liver function were highly correlated with pro-inflammatory cytokines in individuals with alcohol use disorder, which may suggest a possible relationship between chronic alcohol use and presence of a persistent state of inflammation that may eventually increase the risk of comorbid chronic diseases in individuals with alcohol use disorder.Albert et al. present a case study of a 21-year-old woman with daily usage of various synthetic cathinones  who displayed a severe paranoid psychotic state of mind along with a very pronounced psychomotor restlessness which was diagnosed as akathisia during hospitalization. This case study suggests that use of synthetic cathinones may not only increase the risk of developing psychotic-like sequela, but also increase the risk for development of extrapyramidal symptoms such as akathisia, typically observed during use of amphetamine-like psychostimulants. These findings further remark that it is still very difficult to establish a guideline concerning the treatment of intoxication with synthetic cathinones and dependence thereof.Kevin et al. selected 7 synthetic cannabinoid receptor full agonists , representing several distinct chemotypes and toxicological profiles, and evaluated them in a single-point screen against 241 G protein-coupled receptor targets in antagonist and agonist mode using a cellular β-arrestin recruitment assay. The screening yielded few hits in agonist mode for any compound tested aside from CB1 and CB2 receptors, but many hits in antagonist mode, including a range of chemokine receptors, the oxytocin receptor, and histamine receptors. These findings suggest that some “off-targets” could possibly contribute to the toxidrome of synthetic cannabinoid receptor agonists, particularly at high concentrations, and indicate that CBR1-mediated cellular dysfunction may underlie on-target toxicity of synthetic cannabinoid receptor agonists.In conclusion, this Research Topic provides up-to-date information that may enhance our understanding of the central and peripheral toxicity elicited by traditional and emerging drugs of abuse. Importantly, better defining the central and peripheral toxicity for traditional and emerging drugs of abuse could offer understanding on how to derive improved interventive therapeutics and regulatory practices for mitigating drug-driven adverse outcomes.BF, TS, and NS wrote the editorial and approved it for publication. All authors contributed to the article and approved the submitted version.


During transcription, DNA replication and repair, chromatin structure is constantly modified to reveal specific genetic regions and allow access to DNA-interacting enzymes. ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to modify chromatin architecture by repositioning and rearranging nucleosomes. These complexes are defined by a conserved SNF2-like, catalytic ATPase subunit and are divided into four families: CHD, SWI/SNF, ISWI and INO80. ATP-dependent chromatin remodellers are crucial in regulating development and stem cell biology in numerous organs, including the inner ear. In addition, mutations in genes coding for proteins that are part of chromatin remodellers have been implicated in numerous cases of neurosensory deafness. In this review, we describe the composition, structure and functional activity of these complexes and discuss how they contribute to hearing and neurosensory deafness. Eukaryotic chromatin is densely compacted in the nucleus through nucleosomes, an assembly of DNA wrapped around an octamer of two copies of the four histone proteins H2A, H2B, H3 and H4 . ChromatInner ear development is a heavily controlled process governed by dynamic epigenetic regulation, consisting of several mechanisms that ensure the spatiotemporal control of gene expression—one of the fundamental mechanisms to ensure chromatin remodelling. This ATP-dependent mechanism is carried out by a highly evolutionarily conserved set of enzymes belonging to the SNF2 superfamily called chromatin remodellers which are involved in many cellular processes such as transcription, recombination, DNA repair and replication . They arThe inner ear is a multifunctional sensory organ responsible for hearing (cochlea), balance and orientation in space (vestibular system). Following a thickening on the ectoderm surface, the pre-placodal region will give rise to the otic placode at the end of the third week of human gestation. A series of signalling molecules such as FGFs highly regulate this process. The otic placode then invaginates to form the otocyst that goes on to morph into different structures of the inner ear through the modulation of gradients of morphogenic factors. An opposing gradient of Wnt and sonic hedgehog (Shh) defines the dorsoventral axis, a gradient of retinoic acid (RA) defines the anteroposterior axis and at least in part, FGF3 regulates the specification of the mediolateral axis . The aduThe CHD family consists of nine members (CHD1–CHD9). Their structure is characterised by two consecutive chromodomains in the N-terminal region and an SNF2-like ATP-dependent helicase domain positioned in the central region. They recognise and bind nucleosomes to contribute, in many cases, to the formation of heterochromatin typically marked by the presence of methylated histones and other repressive chromatin markers. CHD proteins can bind to methylated histones through their chromodomains and use their helicase activity to remodel the chromatin and contribute to the formation of heterochromatin, a fundamental feature of chromosomes that ensures genomic stability .CHD proteins are classified into three subfamilies (Class I–III) , definedRegardless of overall protein domain structure, the function of CHD superfamily proteins is directly connected to regulating gene expression by controlling chromatin states. They are mainly translational repressors . Here, wCHD3 and CDH4 share 71.6% of their amino acid sequences. Both proteins form distinct nucleosome remodelling deacetylase (NuRD) complexes with different but overlapping functionalities . Mass spCHD3, characterised by intellectual disability, developmental delay  and several dysmorphic features . Th. ThSMARCEach of the two ATPase units SNF2L and SNF2H form heterodimers with either a BAZ-family transcription factor or a larger protein  capable of interacting with acetylated histones . In mammCerc2 have been shown to cause inner ear developmental defects with smaller and wider cochlea and stereocilia disorganisation in hair cells in the organ of Corti [Among these ISWI complexes, only CERF (Cecr2-containing remodelling factor complex) has been shown to play a role in inner ear development and neural tube closure ,100. CERCecr2 on the PCP pathway. Surprisingly, microarray analysis of PCP genes shows that Cecr2 exencephaly is unlikely to directly impact the PCP pathway [The fact that these defects are reminiscent of the misregulation of planar cell polarity (PCP) mutants suggests a possible influence of  pathway . HoweverIt has been shown time and again that ATP-dependent chromatin remodellers are of paramount importance in developmental processes and their involvement in disease, and increasing evidence suggests their implication in the inner ear. Among others, new omics, biophysics and bioinformatics technologies are providing valuable tools to study epigenetic modulation via chromatin remodellers. However, this subject needs to be further investigated in the context of human disease—notably using organoid models, which are rapidly developing, to realistically recapitulate the developmental pathways involved in the emergence of a bona fide inner ear organ, although some challenges remain to be overcome, namely generating actual cochlear-identity hair cells. This would ultimately allow the high-throughput screening of molecules, gene and cell-based therapies, which would accelerate the drug discovery process for diseases involving chromatin remodeller defects.


Mapunda et al.). The current Research Topic brought into attention a diverse panel of original research papers and two reviews on the immune cells involved in CNS pathophysiology, including dendritic cells (DC), mucosal-associated invariant T (MAIT) cells, neutrophils, and pathogenic Th17 cells, as well as a different aspects of that pathophysiology, including molecular signaling pathways (PP2Cδ) and the role of signaling complexes, known as supramolecular organizing centers (SMOCs), therapeutic interventions , possible new forms of autoantigens (neutrophil extracellular traps (NETs) in neuropsychiatric systemic lupus erythematosus - NPSLE), and assessment of correlation between inflammatory markers and severity of autoimmune encephalitis (AE).Insights into the dynamics of immune responses in immune-privileged tissues such as the central nervous system (CNS) are critical to understanding the etiology of autoimmune diseases. Essential in this field is understanding the ways immune cells access and traffic across different types of the blood-brain barrier (BBB), and how the therapeutics alter these processes , it also stimulated histone acetylation, facilitating the expression of genes characterizing hyperactivated DCs. PP2Cδ therefore exerts a key role in DC activation and function via the Rictor/mTORC2/ACLY pathway that controls immune homeostasis via metabolic and epigenetic regulations.It is becoming increasingly clear that metabolism is intrinsically linked to the function of immune cells and their interaction with other cells . DCs repGargano et al. report an evidence of gut dysbiosis of MS patients compared with healthy donors (HD) by metagenomics and metataxonomic analysis of cultivable faecal samples collected from both groups. MAIT cells derived from MS patients show higher level of activation and proliferation in response to fungal isolates overrepresented in MS group , and an increased proinflammatory cytokine production by activated innate immune cells. Importantly, MAIT cells containing pro-inflammatory cytokines were detected in the active brain lesions of MS patients.MAIT cells are highly conserved unconventional T cells activated by riboflavin-derived bacterial and yeast antigens presented by MHC-related protein 1 (MR1). MAIT cells are of explicit interest in multiple sclerosis (MS) , a T-celSim et al. assess the role of LDNs in NPSLE; their proinflammatory function and the participation in the opening of the BBB, hence allowing the anti-neuronal pathogenic antibodies entering the brain parenchyma. The pathogenicity of these antibodies in NPSLE and the role of matrix metalloproteinase-9 and neutrophil gelatinase-associated lipocalin in BBB disruption are also evaluated. Finally, NETs as a source of autoantigens, especially oxidized mitochondrial DNA, enabling to sustain or to initiate autoimmune response, as well as possible therapeutic interventions are discussed.Low density neutrophils (LDNs) were correlated with disease activity in systemic lupus erythematosus (SLE). Their contribution to pathology in SLE is attributed to their capacity to produce type I IFNs and to spontaneously form NETs containing autoantigens and proinflammatory proteins. In the review Mbanefo et al. present a transcription factor STAT3 that plays an important role in the biology of Th1 and particularly Th17 T cells. Although STAT3 is an intracellular protein that is not easily targeted, the authors used the camelid nanobody. In the animal model of experimental autoimmune uveitis (EAU), which targets retina – another immune-privileged tissue, the STAT3-specific antibody was able to efficiently penetrate the blood retina barrier and suppress the differentiation, proliferation, and cytokine release of Th17 cells, leading to EAU amelioration. Authors provide a perspective for the development of future therapeutics to specifically inhibit pathogenic cells, particularly in immune-privileged organs such as the CNS.Infiltration of pathogenic T-cells is a hallmark of several autoimmune diseases including uveitis and multiple sclerosis. Zhang et al. report on a case diagnosed on the basis of rheumatoid factor, anti-cyclic citrullinated peptide antibody and IL-6 positivity in the CSF and the serum together with the unexpected presence of anti-NMDAR Abs in the CSF. Brain MRI confirmed hyperintensity of frontal and parietal lobes. IVIg was effective to alleviate clinical symptoms and lesions and restore normal levels of biochemical indicators except for persisting anti-NMDAR Abs.Rheumatoid meningitis represents a rare complication of rheumatoid arthritis in the CNS. Liu et al. for the first time demonstrated the positive association between MLR and the severity of AE.Autoimmune encephalitis is another inflammatory disease that targets cells of the CNS and causes complex clinical manifestations such as motor dysfunction and psychiatric disorders. Different scoring systems such as the modified Rankin Scale (mRS) or the Clinical Assessment Scale for Autoimmune Encephalitis (CASE) are combined to accurately assess the severity of the disease . In addivia activation of pattern recognition receptors, leading to neuroinflammation and, in later stages, BBB damage and infiltration of peripheral immune cells (Chee and Solito). This neuroinflammation is propagated through SMOCs, comprised of ligands, sensors, adaptors, and effectors that form multiprotein complexes such as myddosomes, inflammasomes, and necrosomes. Sušjan-Leite et al. provide a comprehensive overview of SMOCs involved in neuroinflammation and their role in the development of neurodegenerative and neuroinflammatory diseases by linking innate and adoptive immunity.The increasing incidence of neurodegenerative diseases such as Alzheimer’s is a growing public health problem and a burden on society. Misfolded proteins and aggregates cause direct neurotoxicity and contribute to the production of inflammatory mediators In conclusion, we hope this Research Topic will stimulate new studies testing and expanding therapeutic potentials reported here, dissecting molecular pathways even further to spare immune protection and to provide base for new clinical trials.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the NIH grant 1P01 AI073693-06, Research Fund of The National Institute of Biology (10ICIGEN) and Slovenian Research Agency .We would like to thank all authors for their contributions to this Research Topic. We also thank all the reviewers for their comprehensive and timely evaluation of the submitted manuscripts.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The major histocompatibility complex (MHC) of mammals encodes highly polymorphic classical class I and class II molecules with crucial roles in immune responses, as well as various nonclassical molecules encoded by the MHC and elsewhere in the genome that have a variety of functions. These MHC molecules are supported by antigen processing and peptide loading pathways which are well-understood in mammals. This review considers what has been learned about the MHC, MHC molecules and the supporting pathways in non-mammalian jawed vertebrates. From the initial understanding from work with the chicken MHC, a great deal of diversity in the structure and function has been found. Are there underlying principles? •The genomic organisation of the MHC varies enormously across jawed vertebrates.•Total numbers of MHC genes vary among vertebrates, with only a few classical MHC genes.•Some nonclassical MHC and classical pathway genes appear earlier than others.•Obvious co-evolution within MHC pathways occurs in some species, but not others.•The promiscuity of interactions may correlate with differences in genomic organisation. Current Opinion in Immunology 2022, 77:102218Antigen processingThis review comes from a themed issue on Andrea Sant and Lawrence SternEdited by Antigen Processing (June 2022)”For complete overview of the section, please refer to the article collection, “Available online 7th June 2022https://doi.org/10.1016/j.coi.2022.102218http://creativecommons.org/licenses/by/4.0/).0952-7915/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (The textbook view of major histocompatibility complex (MHC) organisation is that of humans and mice : overallhttps://en.wikipedia.org/wiki/Vertebrate). So far, what have we learned about antigen processing, peptide-loading and antigen presentation from the non-mammalian jawed vertebrates (NMJVs)?Mammals make up only an estimated 5000 mammalian species out of the roughly 66 000 jawed vertebrate (gnathosome) species with MHC molecules  located 5 cM away from the BF-BL region, tapasin located in between class II B genes, two CD1 genes located at the edge of the BF-BL region, and variable numbers of nonclassical class I and class II B genes located in the genetically-unlinked Y region on the same chromosome Xenopus frogs, have a very similar organisation to chickens, including a single classical class I gene next to its APLL genes, a class II gene pair next to their APPL genes, a class III region on the outside and even a region of nonclassical class I genes, the XNCs Overall, the evidence for MHC organisation remains frustratingly fragmented. Some species, like However, other species differ in a variety of ways, highlighting dynamic changes in the genomic organisation in some lineages. The passerines (perching and song birds which make up roughly 50% of bird species) have dispersed their MHC genes over long distances and even on other chromosomes Some fish, like the cartilaginous sharks  have a single MHC, but one or two rounds of genome-wide duplication followed by differential gene silencing have complicated the organisation of the MHC genes in teleosts (the major radiation of ray-finned bony fish). In general, there is a canonical teleost class I region with TAPs, tapasin and inducible proteasome components along with other genes found in the MHC of many jawed vertebrates, with class II and class III region genes spread on different chromosomes 2-microglobulin and the W β2 domain both lack the so-called invariant tryptophan found in nearly all Ig domains, showing the direction of evolution from class II to W to class I genes (For the surviving members of the jawless fish lampreys and hagfish (the earliest fish group to appear in the evolutionary record), there is an adaptive immune system with three kinds of antigen-specific receptors based on leucine-rich repeats, but no genes with obvious similarity to or properties like MHC genes have been reported  I genes . There iThe number of MHC class I and class II genes as well as APPL genes varies considerably among the NMJV, but many species have a single or a dominantly-expressed classical class I gene closely linked to APPL genes, with the remaining class I genes likely to be nonclassical. Classical MHC molecules are defined as those that present peptides to T cells, have high levels of polymorphism and are widely and well-expressed. Nonclassical molecules lack one or more of these features and arise to counter particular threats, appearing and disappearing at different times in evolutionary history, so that some are more ancient and others are present only in particular lineages Xenopus frogs have a single classical class I gene closely linked to the TAP genes, with two lineages of each , along with many nonclassical XNC genes known to be recognised by natural killer T cells Among tetrapods, Xenopus frogs have two allelic lineages with different protease specificities, and the inducible proteasome genes in the bony fish zebrafish have copy number variation There is wide variation in the other APPL genes of the class I pathway. Birds have no inducible proteasome genes, which is surprising since PSMB11 (β5t) is considered essential for positive selection of mammalian thymocytes The situation is even less clear for class II and their APPL genes. In a few species, like parrots, axolotls, Atlantic salmon and medaka, there is only a single classical class II gene pair Outside of mammals, natural ligands have been determined only for chicken classical MHC molecules. A suite of properties was found for the dominantly-expressed class I BF2 molecules which varied in a hierarchy, from quite stringent peptide motifs compared to most human class I alleles to extremely promiscuous In contrast, the BF1 gene is at least ten-fold less well expressed than BF2 and has fewer alleles, many of which have nearly identical peptide-binding motifs A concept of generalist and specialist alleles arose from these findings for BF2 molecules, based on a suite of properties established during peptide loading Xenopus frogs and the bony fish Atlantic salmon may also be promiscuous  and JK was supported by an Investigator Award of the Wellcome Trust (grant number 110106/A/15/Z).RM was supported by Papers of particular interest, published within the period of review, have been highlighted as:• of special interest•• of outstanding interest.


Autism Spectrum Conditions (ASC) are a group of neurodevelopmental disorders characterized by deficits in social communication and interaction as well as repetitive behaviors and restricted range of interests. ASC are complex genetic disorders with moderate to high heritability, and associated with atypical patterns of neural connectivity. Many of the genes implicated in ASC are involved in dendritic spine pruning and spine development, both of which can be mediated by the mammalian target of rapamycin (mTOR) signaling pathway. Consistent with this idea, human postmortem studies have shown increased spine density in ASC compared to controls suggesting that the balance between autophagy and spinogenesis is altered in ASC. However, murine models of ASC have shown inconsistent results for spine morphology, which may underlie functional connectivity. This review seeks to establish the relevance of changes in dendritic spines in ASC using data gathered from rodent models. Using a literature survey, we identify 20 genes that are linked to dendritic spine pruning or development in rodents that are also strongly implicated in ASC in humans. Furthermore, we show that all 20 genes are linked to the mTOR pathway and propose that the mTOR pathway regulating spine dynamics is a potential mechanism underlying the ASC signaling pathway in ASC. We show here that the direction of change in spine density was mostly correlated to the upstream positive or negative regulation of the mTOR pathway and most rodent models of mutant mTOR regulators show increases in immature spines, based on morphological analyses. We further explore the idea that these mutations in these genes result in aberrant social behavior in rodent models that is due to these altered spine dynamics. This review should therefore pave the way for further research on the specific genes outlined, their effect on spine morphology or density with an emphasis on understanding the functional role of these changes in ASC. We do so by delineating supporting correlative evidence in human subjects in Section “Functional Connectivity in ASC Is Linked to Spine Dynamics” and “Many Genes Linked to ASC Are Regulators of mTORC1 or Targets of mTORC1” and use data from rodent models of ASC in further sections in order to explore genetic causality for both spine dynamics and behavior.Autism Spectrum Conditions (ASC) or Autism Spectrum Disorders (ASD) are a group of neurodevelopmental disorders defined by the DSM-VI by the presence of two core symptoms: persistent deficits in social communication, social interaction, socio-emotional reciprocity, and restricted repetitive patterns of behavior, interests, or activities , a set of nuclei that are preferentially activated, as visualized by rsfMRI (resting state magnetic resonance imaging) scanning, while individuals are not focused on the external environment . While DMN activation ceases once cognitive effortful tasks commence  imbalance and reduction in signal-to-noise ratio in neural circuitry that is implicated in ASC , as measured by PET scanning of synaptic glycoprotein 2A, is negatively correlated with symptom severity in adults with MDD, within the dorsolateral prefrontal cortex  haploinsufficiency also reflects the macrocephaly found within ASC individuals  family, made up of mTOR, Raptor, and mLST8  code for the protein hamartin and tuberin respectively which heterodimerize to inhibit RheB, which normally directly activates mTORC1 . In ASC individuals, postmortem studies show a decrease of LC-III, an autophagosome marker, suggesting decreased pruning. Decrease of this marker is correlated with increased spine density and persistent PSD-95 marker expression in the temporal lobe throughout childhood and adolescence  mouse or in the double mutant Tsc2+/−, Atg7cKO mouse, demonstrating an absolute need for spine pruning in order to ameliorate social behavior deficits  and a lack of discrimination in the novel object recognition task. These phenotypes are ameliorated with shRNA to Raptor, an unique component of the mTORC1 complex, suggesting that overactivation of mTORC1 specifically leads to the cognitive deficit  mouse model is sufficient for the manifestation of social preference in the 3-chamber preference test and repetitive behavior in the marble burying task and self-grooming with all behavioral phenotypes ameliorated with rapamycin treatment  KO mice, where mTORC1 is overactive because of loss of inhibition of PI3K, S6K is also overactivated; deletion of S6K increased dendritic spine maturation with concomitant improvement in social interaction behaviors and mGluR-mediated long term depression  ratio, suggesting altered synaptic properties in the CA1 of the hippocampus. They also show decreased vocalization and socialization and no preference for conspecifics in the 3-chamber test as well as increased anxiety and repetitive behaviors such as self-grooming , located on Chromosome 21 respectively have an important role in neuronal development and migration  of this region and whole Shank3 KO displayed reduced sEPSC and LTD that is consistent with a reduced number of glutamatergic synapses on MSN in the striatum , a transcriptional repressor and UBE3A (Ubiquitin-protein ligase E3A), a transcriptional coactivator and E3 ubiquitin ligase that targets proteins for degradation  complex with Drosha and lowers miRNA globally. This includes lowering the miRNA for CREB and LIM kinase (LIMK), whose levels rise; however, dendritic spine density decreases for reasons that are not clear  14 and PND 21 in a Brainbow mouse model results in longer apical dendrite lengths, greater distal arborization and increased soma size  possibly due to an increased proportion of mushroom spines in dentate gyrus granule cells despite similar spine densities as wildtype mice. This accelerated development of glutamatergic synapses can also be seen with persistent propagation of signal in the hippocampal tri-synaptic circuit using photolysis of caged glutamate paired with fast voltage-sensitive dye imaging, demonstrating an increased E/I ratio ”, there are consistent social behavior deficits reported in all rodent models of these upstream regulators of mTORC1 , 4. TestDip2A KO shows lower social exploration time of a conspecific, increased self-grooming in both novel and home cage environments, decreased social novelty and increased marble burying indicative of higher anxiety as well as social recognition and interaction deficits  is decreased and though there is normal social interaction, there is an anxiolytic effect as measured by the elevated plus maze; the molecular mechanisms underlying this phenotype remain obscure  ratio  and FMRP (a negative regulator of mTORC1) reduction reveals a reduction of parvalbumin-positive (PV) GABAergic interneurons such as chandelier and basket calls in the CA1 hippocampus or somatosensory cortex suggesting increased excitability  decrease in spine density and social interaction deficit appears to be NMDA hypofunction since cycloserine, a NMDA agonist, can restore functionality  of the mouse with an increased mEPSC amplitude and frequency in the BLA. This is most likely due to a shift in spine morphology with an increased proportion of mushroom spines but decreased filopodia  which targets PSD-95 for degradation. Hence, in response to neuronal activity in these Fmr1 KO neurons, myocyte enhancer factor (MEF)-induced degradation of PSD-95 degradation and synapse elimination/pruning does not occur leading to higher spine densities  pathways that can activate cortactin and/or LIM kinase (LIMK; Costa et al., via mTORC1. NF-1, a negative regulator of mTORC1, leads to an overall decreased spine density when deleted in neurons, possibly due to inhibition of the ERK pathway which is required for spine stabilization (Ash et al., Amongst negative regulators of mTORC1 such as FMRP, a decrease in phosphorylated active SLINGSHOT protein, an inhibitor of LIMK combined with an increased phospho-LIMK due to increased Rac/PAK levels, leads to a decreased level of active cofilin and subsequent actin depolymerization (Pyronneau et al., Met, a tyrosine kinase that regulates spinogenesis, demonstrates local hyperconnectivity in this circuit with stronger afferents from corticostriatal neurons in layer 2/3 to layer 5 projection neurons in the anterior frontal cortex in heterozygotes and KOs compared to WT controls (Qiu et al., Corticostriatal circuitry which underlies motivated sensorimotor by interacting with the limbic system is the focus of several neurodevelopmental disorders, including autism (Shepherd, Shank3, a positive regulator of mTORC1, show that the striatal excitability is higher due to increased cortical glutamatergic efferents to the striatum (Peixoto et al., Tsc2+/− mice, resting state fMRI reveals functional hyperconnectivity within the corticostriatal circuit due to increased synaptic coupling possibly as a result of increased spine density; this was strongly correlated to repetitive behaviors. All parameters, i.e., connectivity, spine density, synaptic coupling, resting state default mode network signatures, and social behaviors are rescued to normal levels with rapamycin, suggesting that mTORC1 overactivation in this circuit is crucial to spine dynamics that underlie social behaviors. This hyperconnectivity signature is valid cross-species—children with ASC showed similar fronto-insular-striatal hyperconnectivity and mTORC1 interacting genes are enriched in the ASC cortical transcriptome, suggesting that this Tsc-mTORC1 pathway is a critical driver of functional hyperconnectivity (Pagani et al., Tsc1/2, though Nf1 KO mice show decreased spine density. Children with NF1 mutations with ASC show reduced functional connectivity between the striatum and the frontoparietal network and increased striatal functional connectivity with the limbic system. Similar to the human scenario, Nf1+/− mice also show disrupted corticostriatal connectivity, as revealed by resting state imaging (Shofty et al., Developmental studies of Despite a number of studies that show both spine dynamics and social behavior alterations in ASC rodent models, a number of questions persist. Many models are homozygous deletion models in order to obtain more robust effects whereas they tend to be haploinsufficient conditions in the human (Monteiro and Feng, Furthermore, though several of the upstream regulators such as SynGAP and SHANK have isoforms, the differential contribution of isoforms is not clear and deserves more attention. For example, SynGAPα1 is the only isoform capable of being anchored at the PSD due to the presence of its unique C-terminal PDZ binding domain and is sufficient for the enlargement of spine heads by AMPA receptor insertion upon chemically induced LTP as well as increase in PSD-95-positive puncta. SynGAPβ isoform rescues the reduction in dendritic arborization seen in cultured hippocampal neurons when SynGAP levels are reduced (Araki et al., Shank3−/− mice, mGluR5 is increased in the striatum but not in the cortex (Wang et al., Shank3 mice deleted for the ankyrin repeat domain show reduced excitation but increased frequency of sIPSCs at Schaffer collateral synapses in pyramidal neurons in the hippocampus. However, prelimbic layer 2/3 pyramidal neurons in the medial prefrontal cortex show decreased frequency of sIPSCs, though the basis of this difference is not known (Lee et al., Shank2 PDZ-deletion mutant mice, NMDA hypofunction also leads to lower LTD and lower LTP at Schaffer collateral synapses but not in the prefrontal cortex (Won et al., Tsc1 mutant (Dalal et al., +/− mice, there was increased expression of FMRP targets (Hien et al., Tsc-Fmr1 regulation in the cerebellum vs. the hippocampus. Apart from the hippocampus, cortex and cerebellum, many social behaviors are regulated by nuclei in the social behavior network which includes hypothalamic nuclei (Newman, In addition to isoform-specific functions of mTORC1 upstream regulators, the role of these genes has typically been studied in two major brain regions, i.e., the hippocampus and cortex and region-specific differences exist . For exa Newman,  in a sex Newman, , this woThough the picture for changes in spine dynamics upon deletion or duplication of these genes is complex, it can be simplified when seen through the lens of regulation of mTORC1 as part of a larger grouping  and 4; tNV framed and analyzed most of the scientific content in the manuscript. SC did the analyses of individual mTORC1 regulators including the summary of the spine density and morphology in tabular form. Both SC and NV discussed and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TBX20 gene has a key role during cardiogenesis, and it has been related to epigenetic mechanisms in congenital heart disease (CHD). The purpose of this study was to assess the association between DNA methylation status and congenital septal defects. The DNA methylation of seven CpG sites in the TBX20 gene promoter was analyzed through pyrosequencing as a quantitative method in 48 patients with congenital septal defects and 104 individuals with patent ductus arteriosus (PDA). The average methylation was higher in patients than in PDA (p < 0.001). High methylation levels were associated with a higher risk of congenital septal defects . The ROC curve analysis indicated that methylation of the TBX20 gene could be considered a risk marker for congenital septal defects . The analysis of environmental risk factors in patients with septal defects and PDA showed an association between the consumption of vitamins  and maternal infections . These results suggest that differences in DNA methylation of the TBX20 gene can be associated with septal defects.The  NKX2.5, GATA4, TBX1, TBX5, TBX20, LINE1, and HAND1 ; the comparison group included 104 non-syndromic PDA paediatric individuals  with a median age of 3 years [IQR: 2–6]. These general characteristics of the study population are presented in p < 0.05). The other factors evaluated  showed no significant differences. The specific environmental risk factors analyzed in mothers of individuals with CHD are represented in The presence of environmental risk factors during pregnancy in mothers of individuals with CHD showed significant differences in the consumption of vitamins, maternal infections, and medication consumption  in the highest quartiles of the average of all CpG sites. Afterward, the model was adjusted for maternal infections, and consumption of vitamins the risk association remained . The association data of methylation levels of each CpG site showed similarly a significantly increased risk of septal defects development in the highest quartile; CpG site 1: ; CpG site 2: ; CpG site 3: ; CpG site 5: ; CpG site 6: ; and CpG site 7: ; with the exception of CpG site 4: . Data are described in The stratification of DNA methylation levels of the study groups into quartiles allowed us to identify a significant risk of septal defects development , The ROC curve analysis showed an AUC of DNA methylation levels of p = 0.048) of the disease, while maternal infections were associated with an increased risk of septal defects . Medication consumption showed a trend toward an increased risk; however, this was not statistically significant. The other factors did not show any association  (Based on the quartiles of ctively) . As the bHLH) and Specificity protein 1 (Sp1) transcription factors.The in silico analysis through bioinformatic tools showed binding sites for Hey-like basic Helix-Loop-Helix transcription factors  biological process: the cardiac chamber formation is marked in blue, and the atrial septum formation is marked in red; (b) disease gene associations: the atrial septal defect is marked in green and the ventricular septal defect is marked in yellow .TBX20 gene promoter and age by study groups, and the result showed a moderate positive correlation in the septal defect group as well as in the PDA group  (p = 0.013) but not in male patients with septal defects . Similarly, a positive correlation was found in females and in males  of the PDA group.  . LikewisA group. .p < 0.001]; nonetheless, the comparison between male patients with septal defects and the male PDA group did not show a significant difference  (p < 0.05); when males with septal defects were compared to PDA males, no significant difference was observed (p > 0.05). Data are described in A significant difference was found between the median value of average all sites of DNA methylation levels in female patients with septal defects and the female PDA group  . The CpGDuring the cardiogenesis process, the participation of several transcription factors in heart formation is reported . In the TBX20 gene promoter, influencing the development of septal defects. In addition, the analysis of environmental risk factors suggests a possible relation between the consumption of vitamins and maternal infections with septal defects. The analysis of interaction using MDR showed models suggesting an interaction between TBX20 methylation, exposure to pollutants, maternal infections, sex, and age. In this study, we found evidence of an epigenetic mechanism, DNA methylation of bHLH and Sp1 transcription factors, as well as a PPI network, where the TBX20 protein interacts with proteins encoded by cardiac genes, therefore, exhibiting an important role in the biological processes involved in the heart formation and gene-associated diseases such as ventricular and septal defects. On the other hand, the results of the bioinformatics analysis showed binding sites for TBX20 is one of the primary genes involved in heart development; to date, however, its epigenetic mechanisms in CHD are not fully understood. During heart formation, the TBX20 gene has a key role in the regulation and proliferation of cardiac cells, as well as in the formation of cardiac chambers [chambers ,35. Thischambers ,13,14.TBX20 gene in patients with ventricular septal defects (VSD) and found higher methylation levels in the patient group than in the control group [We observed high DNA methylation levels in patients with septal defects compared to the PDA group. These findings are in accordance with the study by González-Peña et al.; they evaluated the methylation status of the ol group . That stTBX20 gene promoter, we found a similar pattern of methylation with higher levels in the septal defects groups than in the comparison groups; therefore, the region near the transcription start site could represent an important regulator of transcriptional activity of TBX20 gene [TBX20 gene in the development of CHD. Our study, on the other hand, included a larger number of individuals in each group; additionally, we evaluated several environmental risk factors associated with CHD, while González-Peña et al. focused on the maternal dietary intake of folic acid. It is important to note that the experimental strategy used in both studies to determine the methylation levels were different. We used the pyrosequencing process, and González-Peña et al. used the Quantitative PCR (qPCR). Although both studies analyzed different regions of the X20 gene . To confTBX20 gene in other types of CHD, specifically in tetralogy of Fallot (TOF). TOF is caused by a single initial alteration, consisting of hypoplasia of the infundibular septum, characterized by interventricular communication, obstruction of the right ventricular outflow tract override of the ventricular septum by the aortic root, and right ventricular hypertrophy [TBX20 gene in eight patients with TOF and five controls and reported higher methylation levels in controls [TBX20 gene in 23 patients with TOF and 5 controls, finding lower methylation levels in TOF [Previous studies have evaluated the methylation status of the ertrophy ,38. In tcontrols . Yang ets in TOF . Additios in TOF . In brieTBX20 gene promoter. These results suggest that high DNA methylation levels of the TBX20 gene could be a good strategy for assessing the risk of septal defects.The ROC curve analysis showed a discriminatory capacity to predict the presence of septal defects by analyzing the methylation levels of the TBX20 gene [In addition, we identified differences in methylation levels that might be associated with septal defect development. The methylation status in the promoter region could lead to transcriptional repression and disrupt the gene expression and function of the X20 gene ,42. ExpoX20 gene ; regardiX20 gene .Our analysis of environmental risk factors suggests that the consumption of vitamins, such as folic acid and ferrous fumarate, could be protective factors against septal defects. This agrees with the literature: in a meta-analysis, Feng et al. evaluated epidemiological observational studies of folic acid consumed by pregnant women before becoming pregnant and the risk of CHDs in their offspring, finding robust evidence that maternal folic acid (FA) intake reduces the risk of CHDs . SimilarTBX20 gene associated with VSD, reporting that a higher methylation status of the TBX20 gene was associated with FA maternal intake [Regarding maternal intake of FA, González-Peña et al. also evaluated mothers of patients with VSD and a control group. They found a relationship between maternal intake FA and the methylation status of the l intake . On the l intake . Similarl intake . NeverthTBX20 methylation, exposure to pollutants, age, and sex, which could be evidence of a necessary interplay of environmental factors with epigenetic mechanisms, such as DNA methylation, in the development of CHD [The association of environmental factors with CHD agrees with previous reports; however, nowadays, the molecular mechanisms of regulation of these environmental factors in the etiology of CHD are not clear. In our study, the MDR analysis identified a significant interaction between t of CHD ,50,51,52TBX20 methylation with age. This result agrees with the finding of a correlation between methylation levels of TBX20 and age since we observed a moderate increase in methylation levels according to age in the septal defect group as well as in the PDA group. Previous studies have described this phenomenon, indicating that the CpG sites distributed along the whole human genome could present dynamic changes according to age [Additionally, the MDR analysis showed an interaction between g to age ,54,55. NbHLH) transcription factor and Specificity protein 1 (Sp1). bHLH transcription factors are transcriptional repressors, while Hey proteins participate in cardiomyocyte differentiation [TBX20 gene; however, this hypothesis should be validated in functional studies with another regulatory mechanism to confirm these findings. The bioinformatics analysis to identify binding sites in the analyzed sequence showed binding sites for Hey-like basic Helix-Loop-Helix (ntiation  and reguntiation . It has ntiation . Sakata ntiation . Our finSp1 is a transcription factor member of the Sp/Kruppel-like factor (KLF) family, having a fundamental role in different biological processes, with a relevant function in embryonic development, cell cycle regulation, and cell differentiation; Sp1 has been implicated in several diseases [Sp1 presents binding sites for GC- or GT-boxes. It has the capacity to promote and activate transcription in the promoter region of eukaryotes [diseases ,61. Sp1 karyotes ,63 and tkaryotes .Sp1 in TBX5 gene promoter analyzed by pyrosequencing in patients with septal defects [TBX20 gene in patients with Tetralogy of Fallot and found a hypomethylation in the TBX20 gene promoter region in the patient group. They reported that high expression of the TBX20 gene could be caused by Sp1 binding at the region analyzed due to the decreased methylation at the Sp1 transcription factor binding sites [Nkx2.5 knock-out embryonic mice and constructed a transcriptional regulatory network to identify differentially expressed genes (DEGs) and reported a down-regulation of Sp1 among other transcription factors; they concluded that DEGs could provide new knowledge to understand the mechanisms implicated in the development of CHDs [Sp1 having a possible key role in regulating the transcription of genes that participate in the pathogenesis of CHDs. This factor has been reported previously in CHDs. García-Flores et al. observed binding sites for  defects . Gong J ng sites . Li et a of CHDs . These rNkx2.5, GATA4, GATA6, HAND2, and ILS1) that are associated with heart formation and CHDs [Thus, the use of bioinformatics tools is an excellent strategy for performing analysis by computer simulations and providing experimental data to explain the mechanisms involved in different diseases. In this study, the protein-protein interaction network for the TBX20 protein identified, in the functional enrichments analysis, its interaction with several transcription factors  we evaluated the DNA methylation levels by a quantitative experimental strategy: pyrosequencing, (c) we used bioinformatic tools to perform in silico analyses, where the computational modeling included the prediction of bindings sites for transcription factors and the development of protein-protein interaction network for the TBX20 protein.Our study has several strengths: (a) we assessed the effect of being exposed to different environmental risk factors during pregnancy in mothers of individuals with CHDs on the methylation levels of TBX20 gene, (c) we could not perform a genetic investigation such as array-CGH or NGS panels to discard the presence of mutations in genes associated with CHD; nevertheless, this assessment was conducted by clinical and phenotypic examination performed by two experienced medical geneticists who discarded any syndromic association, d) we have a limited number of participants. Thus, additional studies in other populations with different genetic backgrounds and with larger samples of CHD are required to confirm our findings.Our study also has some limitations that should be mentioned: (a) we analyzed a region belonging to one CpG Island, additional CpG Islands should be evaluated in order to identify the methylation status of the promoter region, (b) we did not perform a functional study to validate the effect of the methylation status on the regulation of transcriptional activity of TBX20 gene promoter are associated with congenital septal defects. Moreover, we identified some environmental factors that can be used as protective and risk markers for developing congenital heart defects. These findings contribute to new knowledge in the epigenetic field involved in the pathogenesis of congenital septal defects. Further studies should be performed to confirm our findings.Our findings suggest that high DNA methylation levels of the