Glutathione (GSH) is the most abundant non-protein thiol present at millimolar concentrations in mammalian tissues. As an important intracellular antioxidant, it acts as a regulator of cellular redox state protecting cells from damage caused by lipid peroxides, reactive oxygen and nitrogen species, and xenobiotics. Recent studies have highlighted the importance of GSH in key signal transduction reactions as a controller of cell differentiation, proliferation, apoptosis, ferroptosis and immune function. Molecular changes in the GSH antioxidant system and disturbances in GSH homeostasis have been implicated in tumor initiation, progression, and treatment response. Hence, GSH has both protective and pathogenic roles. Although in healthy cells it is crucial for the removal and detoxification of carcinogens, elevated GSH levels in tumor cells are associated with tumor progression and increased resistance to chemotherapeutic drugs. Recently, several novel therapies have been developed to target the GSH antioxidant system in tumors as a means for increased response and decreased drug resistance. In this comprehensive review we explore mechanisms of GSH functionalities and different therapeutic approaches that either target GSH directly, indirectly or use GSH-based prodrugs. Consideration is also given to the computational methods used to describe GSH related processes for in silico testing of treatment effects. During the complex and tightly regulated oxidation processes such as glycolysis in the cytosol, and citric acid cycle and oxidative phosphorylation reactions in the mitochondria, electrons are used to power the synthesis of ATP, which then supports the high energy demands of tumor cells. However, during fuel oxidation, electrons can escape and interact with oxygen to form ROS. Mitochondrial redox reactions are a major source of ROS in most cell types, including tumor cells. ROS are generated by the NADPH oxidase (NOX) enzymes, located on the plasma membrane of cells, notably in neutrophils and vascular endothelial cells. In order to avoid the toxic effects of ROS on DNA, proteins, and lipids, cells have evolved a wide array of antioxidant mechanisms, and this is particularly true in tumor cells where the activities of redox reactions are extremely high. Many of the ROS defense mechanisms depend on the tripeptide molecule, GSH. Tumors also have an increased burden of reactive nitrogen species (RNS) which are generated enzymatically by nitric oxide synthases (NOS), located in tumor cells, stromal cells or infiltrating immune cells. NOS activity produces NO gas that reacts non-enzymatically with cysteine residues in glutathione, major non-protein thiol and protein-thiols to form S-nitrosoglutathione (GSNO) and S-nitrosothiols in a process known as S-nitrosation. Through these reactions as well as its interactions with a number of proteins, glutathione is one of the major regulators of cancer cell development, progression, response to therapy and its environment leading to a number of avenues for therapeutic targeting  metabolism, which is now known to extend beyond its well-known central role in the regulation of reactive oxygen species (ROS). While ROS are inevitable byproducts of oxidative metabolism, their production and neutralization are controlled by an extensive array of processes. ROS are inextricably linked to the cellular oxidation of fuels, when liberated electrons  modify the balance of reduced and oxidized pairs of electron acceptors such as NADH/NADargeting .In this review, we focus on the role of GSH in cancer cell metabolism, tumor microenvironment and communication leading to its function in tumor progression and treatment.−2), hydrogen peroxide (H2O2) and/or hydroxyl radical (OH). Superoxide anion has a short half-life and is quickly converted to hydrogen peroxide by the superoxide dismutase enzymes, SOD1 and SOD2, located in the cytoplasm and mitochondria, respectively. In comparison to superoxide, hydrogen peroxide is much longer-lived  [2O2 can react with ferrous ions in a reaction known as the Fenton reaction to generate the short-lived hydroxyl radicals, which are highly toxic and can cause irreversible damage to essentially all types of cellular macromolecules, including nucleic acids , lipids , and proteins  [−), which can cause nitration of protein tyrosine residues thereby affecting protein function [The major known forms of ROS include superoxide anion radical O−, hydrogeylation) ,4,5. Sup+ [In mitochondria the major sources of ROS include OXPHOS complexes I and III, and the dehydrogenases that use ubiquinone as an acceptor, and this includes succinate dehydrogenase (OXPHOS complex II). Other distinct mitochondrial sites associated with substrate oxidation and oxidative phosphorylation leak electrons to oxygen to produce superoxide or hydrogen peroxide, and these include oxoacid dehydrogenase complexes that shuttle electrons to NAD+ . Mitocho+ . This is+ ,9. Recen+ .2O2 [2O2 can oxidize these residues, leading to their sulfenylation (S-OH), sulfenamidylation (S-N), glutathionylation (S-SG), or disulfide bond formation between protein cysteine residues (S-S). All of these can modify the structure and activity of proteins [2O2 exposure can cause thiol sulfinylation (SO2H) and sulfonylation (SO3H), which irreversibly damage the protein.Nonetheless, it is currently well recognized that low levels of ROS are important for many types of cell signaling reactions; this is especially true for the longer lived H2O2 . One of proteins . These pA battery of mechanisms exists in cells to keep ROS levels within the non-toxic range, and these mechanisms are exquisitely well regulated to simultaneously allow low levels of ROS to act as signaling molecules. This is particularly relevant in tumor cells, as described below.2O2); catalases (converts H2O2 to H2O and O2); and glutathione peroxidases (converts H2O2 to 2 molecules of H2O). Conversely, the hydroxyl radical cannot be detoxified enzymatically, contributing to its detrimental effects in cells.The well-recognized enzymes directly neutralizing ROS include the SODs (converts superoxide to H2O2. GSH is directly used in glutathione-S-transferase (GST) reactions in the detoxification of xenobiotics or products of oxidative stress [2O2 production to NNT-associated NADPH and glutathione redox mechanisms [2O2 and the peroxiredoxins (PRXs) and glutathione peroxidases (GPXs) to convert H2O2 to H2O.GSH is often referred to as the most important non-enzymatic antioxidant in cells, as it is essential in various antioxidant processes. GSH is found throughout cellular compartments in millimolar concentrations (1–10 mM). GSH contributes to the maintenance of ‘healthy’ levels of ROS in several ways . First, e stress . The reve stress ,14. In ae stress . As GSH e stress . The mitchanisms . Beyond chanisms ,19. AltoThe widely dispersed cellular localizations of the various isoforms of these enzymes normally protect cells very well from oxidative damage. Relevant to the central role of GSH, glutathione peroxidase 4 (GPX4) plays an additional protective role in catalyzing the reduction of organic hydroperoxides, and lipid peroxides in membranes. Finally, under oxidized redox potential conditions, the oxidized form of GSH can reversibly bind to protein thiol residues to protect them from irreversible oxidative damage, and/or modify their activity. Some of our previous research demonstrated that the glutathionylation of the mitochondrial uncoupling proteins UCP2 and UCP3 contributed to the control of mitochondrial ROS levels ,20,21,22One important example of reactive nitrogen species (RNS), is NO, a reactive gas produced by a family of enzymes known as nitric oxide synthases (NOS), namely endothelial NOS, neuronal NOS and inducible/macrophage NOS. Due to the high concentration of GSH in the cells, NO rapidly reacts with the cysteine thiol group of GSH to form S-nitrosoglutatione (GSNO). GSNO represents a reservoir of NO bioactivity and a delivery system of NO to maintain cellular homeostasis . As a scNO can also react with sulfhydryl moieties of cysteines in proteins to form S-nitrosoproteins or S-nitrosothiols in a process known as S-nitrosation, which results in a change in the activity and function of proteins. Similar to other redox signaling mechanisms such as phosphorylation, acetylation and ubiquitylation, S-nitrosation has emerged as an important redox-based post-translational modification of proteins ,28. A reRedox and ROS are also of particular importance in tumor cell signaling pathways associated with cancer progression through their roles in tumorigenesis, tumor cell migration, and tumor cell survival ,30,31. CRAS, and oncogenic K-RAS have been found to be necessary for precancerous lesions in the pancreas and for lung adenocarcinoma formation [Tumor hypoxia is well known to drive higher ROS production . The higormation ,36.ND6 mutations leads to high levels of ROS generation and a highly metastatic phenotype that can be prevented by ROS scavengers [ND5 mutation that results in apoptosis resistance and increased activity of the PI3K/Akt pathway [In many cancers associated with mutations in genes encoding electron transport chain proteins there is increased ROS production that is associated with resistance to apoptosis. For instance, impaired Complex I function associated with avengers . As well pathway . There i pathway . Thus, tThe increased ROS levels in some tumor cells are also commonly associated with decreased expression of cellular antioxidant systems. For example, the loss of tumor suppressors such as p53 can contribute to tumorigenesis through the elevated ROS levels occurring because of the cellular antioxidant systems’ loss. p53 increases the expression of SOD2 and GPX1 as well as NADPH production ,40,41.However, the increased production of ROS by many tumor cells occurs concomitantly with increased expression and activity of antioxidant systems. This may be related to the findings that high levels of ROS can mitigate metastasis and tumorigenesis, for example in cancer cells detaching from the extracellular matrix ,43. IndeElevated levels of GSH have been observed in many cancers including breast, ovarian, lung, as well as head and neck cancers . ThroughThe observed increase in GSH levels, is due not only to the higher levels of ROS production in most tumor cells, but it is again related to the fact that some of the classical tumor promoters also activate GSH synthesis and turnover mechanisms  . In tumo2+ as a cofactor are required to facilitate this reaction. Furthermore, the GCL enzyme is composed of a catalytic and modifier subunit ; GCLM is subject to feedback inhibition by GSH. The second and final step of the pathway produces GSH via the addition of glycine to the γ-glutamylcysteine intermediate. [GSH biosynthesis is a well-conserved pathway in which the three precursor amino acids, cysteine, glutamate, and glycine, are combined to form the tripeptide GSH. In animals, this process occurs exclusively in the cytosol, where the two enzymes glutamate cysteine ligase (GCL) and glutathione synthetase (GS) are localized. GCL catalyzes the first, and rate-controlling, step of the pathway that creates a linkage between glutamate and cysteine via the γ-carboxyl residue of the latter to produce γ-glutamylcysteine . ATP hydmediate. . Coupledmediate. . These pIt is well-known that shifts in GSH metabolism often accompany tumor development, potentially as a way for the cancer cells to mitigate the impact of increased oxidative stress that results from their extraordinary metabolic rates . For exaMetabolic heterogeneity, resulting from factors both intrinsic and extrinsic to the cancer cell, leads to tumor subtype specific metabolic properties and vulnerabilities . DiversiThe range of functions of GSH illustrate the great demand for this molecule not only throughout the cell and its organelles, but also in the surrounding extracellular environment. Due to the properties of GSH (namely its size and net negative charge), GSH transport generally requires some form of carrier to cross the plasma membrane, or the membranes of organelles to reach its many targets. Additionally, GSH is not only the most prominent non-protein thiol in cells, it is also one of the most abundant metabolites, which further illustrates the demand for transport . UnfortuSome of the earliest investigations into the membrane transport of GSH came with the transporter, multidrug resistance protein 1 (MRP1). The MRP or ABCC family of proteins are a group of transporters found on the plasma membranes of many cells. Early studies found that MRP1 could provide tumor cells with multidrug resistance (MDR) by exporting xenobiotics, and that this function was associated with GSH . The detSeveral organelles have been demonstrated to require their own GSH pools, including the nucleus, endoplasmic reticulum, and mitochondria. These organelles generally have similar concentrations of GSH as in the cytosol, although their redox ratio (reduced GSH to oxidized GSSG) may vary . MitochoDue to its role in DNA repair and protection from oxidative damage, the nucleus seems like an obvious target for GSH localization. Indeed, the antioxidant plays an important role within the nucleus and in cell proliferation ,76. InteCordyceps militaris, may be a promising anticancer therapy via regulation of GSH [Unlike in other organelles, investigations into the mechanisms behind endoplasmic reticulum (ER) GSH transport have been more successful in identifying the proteins responsible. The channel protein Sec61 has been demonstrated to import and export ER GSH, and transport is modulated by Bip and Ero1 . Interesn of GSH . In the Another important GSH pathway is the antioxidant pathway via the mitochondrial enzyme GPX4, which is responsible for the elimination of damaging lipid peroxides, and other ROS species. This pathway is of particular relevance to disease as oxidative stress is a hallmark of many cancers, neurodegenerative disease, and more; one result of this stress being lipid oxidation . MoreoveFurthermore, GPX4 is an essential enzyme in the recently discovered ferroptosis cell death pathway. A major result of this is the depletion of GSH, and disruption of the GPX4 antioxidant pathway, leading to massive lipid peroxidation and cell death . GPX4 isAs mentioned above, cellular damage may arise from small molecules including metals such as iron and copper. Protecting against these, and other, potentially toxic species is another challenge faced by the cell. A superfamily of enzymes known as glutathione transferases (GSTs) utilize GSH to combat a wide range of xenobiotics and other harmful compounds . These eMost relevant of these are the previously mentioned xenobiotic reactions, in which GSTs function as Phase II detoxification enzymes to eliminate a variety of hydrophobic and electrophilic compounds via GSH ,67. The Within cancer, GSTs play a significant role in both tumorigenesis, as well as in established cancer cells. Interestingly, in tumorigenesis these enzymes protect from cancer development but once this fails GSTs are essential for ensuring the survival of tumor cells . For exaGSTs have been linked to MDR in tumors, a common problem in treatment of many cancers. Members of the super family have long been known to be overexpressed in many types of cancers ,96,97. AChronic infection, injury and inflammation have long been recognized as risk factors for the development and progression of various human cancers . There i2O2, organic hydroperoxides and peroxynitrite [PRDX is a family of ubiquitously expressed proteins that catalyzes reduction of Hynitrite . In humaynitrite  with a nynitrite , lung [1ynitrite  or pancrynitrite . In addiInflammatory stimuli cause release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme that extracellularly acts as a redox-dependent inflammatory mediator. The glutathionylation of GSH to PRDX2 cysteine residues has been shown to be central to the regulation of immunity. Additionally, several studies show presence of PRDXs in body fluids of cancer patients, suggesting that PRDXs and its glutathionylation may be a link between inflammation and cancer . Additio2O2, organic hydroperoxides and/or lipid hydroperoxides to generate their corresponding lipid alcohols and water at the expense of oxidation of two molecules of GSH [Tumor cells have increased ROS levels and thus upregulate the antioxidant response, including glutathione peroxidases (GPXs). GPXs are members of a multi-enzyme family that catalyze the reduction of Hs of GSH . There aGPX genes have been utilized to study the potential role of individual GPXs in malignancy. Several in-depth reviews have been published on the molecular mechanisms of GPXs regulation in tumors and their function in different stages of tumor progression [GPX1 and GPX2 genes, spontaneously developed ileocolitis at a young age. The ileum of these mice lost GSH-dependent activity to reduce peroxides and had increased expression of NOX1 mRNA and elevated levels of TNF, suggesting that NOX1 is the major source of ROS and further supporting the anti-inflammatory function of GPX1 and GPX2 [Cellular models and transgenic mice deficient in individual gression ,116,117.gression . For insand GPX2 . Using Gand GPX2 . Taken tGPX3 is the only extracellular GPX that detoxifies lipid hydroperoxides at the expense of GSH. GPX3 mRNA is expressed in the kidney, pancreas, lung, breast, brain and gastrointestinal tract, but the majority of GPX3 is expressed in the plasma which is kidney-derived. It has been reported that GPX3 can act both as a tumor suppressor and a pro-survival protein. Lower GPX3 levels in the plasma and tumor tissue of non-small-cell lung cancer, glioblastoma, hepatocellular carcinoma and colorectal carcinoma have been linked to selenium deficiency and increased lipid peroxidation, supporting the notion that loss of GPX3 contributes to oxidative stress . GPX3 loThere are also studies demonstrating that GPX3 expression is increased in certain cancers, including clear cell adenocarcinoma, colorectal carcinoma, ovarian cancer and leukemia . Hence tGPX4 in cells and mice resulted in increased lipid-based oxidative stress and induction of 12/15-lipoxygenase, lipid peroxidation and apoptosis-inducing factor-mediated cell death [GPX4 is also an important enzyme that protects membranes against lipid peroxidation . It has ll death . GPX4 dall death . While t2+ breast tumors as well as resistance to trastuzumab [NO is produced enzymatically by nitric oxide synthases (NOS) not only by tumor cells but also by infiltrating inflammatory immune cells and stromal cells ,126. As stuzumab . More restuzumab . TherefoSolid tumors can be considered as complex organs composed of not only tumor cells but also a diverse population of non-tumor cells that form the physical components of the tumor microenvironment (TME). The non-tumor cells include stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs), tumor vasculature consisting of endothelial cells and pericytes as well as infiltrating immune cells consisting of lymphocytes, neutrophils, macrophages, and mast cells. The physiological characteristics of the TME are defined by its pH and oxygen tension as well as presence of nutrients, cytokines, chemokines, growth factors, matrix remodeling enzymes, and metabolites . Tumors Exosomes are nanometer-sized (~40–150 nm) membrane-enclosed vesicles that are derived from the endosomes through inward budding to generate multivesicular bodies (MVB). The MVB fuse with the plasma membrane to release EVs in the TME. It has now become clear that essentially all cell types constitutively secrete exosomes into the extracellular space to mediate intercellular communication . Within Over the past decade, research efforts in tumor biology have highlighted the pivotal role of exosomal oncogenic proteins and RNAs to modulate the TME and support tumor progression ,143,144.Recent studies have revealed that CAF-derived exosomes in the TME can facilitate communication between tumor and stromal cells and contribute significantly to tumor progression ,148. TheTumor hypoxia and acidosis have emerged as important physiological factors that can affect the redox status, including elevated concentrations of GSH in the TME ,152,153.Following cancer onset, glutathione’s antioxidant function can become superfluous in some cancer subtypes due to thioredoxin antioxidant pathway fulfilling the antioxidant requirements making inhibition of only GSH insufficient for tumor therapy . HoweverChemotherapy treatment of triple negative breast cancer leads to an increase in expression of GCLM and SLC7A11 and an increase in the intracellular GSH concentration. GSH functions as a signaling molecule which activates the breast cancer stem cell phenotype through copper chelation that leads to an inhibition of mitogen-activated protein kinase kinase (MEK) activity, leading to inactivation of MEK1-ERK signaling, FoxO3 dephosphorylation, and nuclear translocation ultimately leading to enrichment of breast cancer stem cells High ROS production in cancer cells requires high activity of cellular antioxidant systems making cancer cells hypersensitive to exogenous agents that damage their antioxidant capability. Therefore, reduction in either glutathione production or availability provides an avenue for tumor therapy. Depletion of GSH in cells ultimately leads to ferroptosis .GSH acts as a detoxifying agent by direct conjugation to a xenobiotic (X) forming a glutathione-S conjugate as part of the mercapturic acid pathway that leads to the elimination of toxic compounds. In cancer cells this process can be used for the elimination of chemotherapeutic drugs.GSH synthesis is primarily controlled by the availability of cysteine, with cysteine starvation leading to GSH depletion. Therefore, inhibition of the cysteine cell transporter system xc− (cystine/glutamate antiporter protein SLC7A11) can lead to GSH depletion and cell death through ferroptosis. This approach is explored with small molecule drug erastin (named after eradicator of RAS and ST-expressing cells) identified to efficiently kill human tumor cells without affecting their isogenic normal cell counterparts . AdditioMany cancer drugs exhibit high toxicity making target selectivity a major focus for cancer therapy development. High concentration of GSH in many tumors together with its high reactivity leads towards utilization of GSH as an activator of prodrugs, possibly leading to GSH depletion as well as release of toxic chemotherapeutic agents. An example was presented by Grant et al.,  with romDevelopment of ROS and GSH responsive nanoparticle-drug delivery systems has been proposed as a way to more specifically and safely deliver highly toxic payload to cancer cells . An examAlternatively, prodrugs activated by other processes specific to cancer cells can be used for the depletion of GSH. Quinone methide (QM) is an intermediate produced by esterases enzymes and is capable of depleting GSH in cells. As esterases are overexpressed in many cancer subtypes utilization of prodrug esters that are “activated” by the function of esterases into QM to perform GSH depletion is very promising with a number of publications presenting different molecular constructs for prodrug. A recent example of this approach Yoo et al.  has presSelection of GSH related tumor therapies from analysis of only a subset of its functions can result in highly unexpected results. Computer modeling provides a platform for combining, testing, and predicting glutathione’s many roles as well as testing different routes for its regulation through synthesis, transport, or availability of amino acid components. Computer modeling is an integral process of drug discovery from analysis of optimal targets from large omics data, to modeling, docking or AI driven drug design to modeling of cellular responses to therapy. In cancer applications computer modeling can be used to establish a role of glutathione within cancer metabolism and for providing in silico routines for testing effects of drugs and relevance of drug targets. Mechanistic or machine learning modeling of processes as well as modeling of molecular interactions can be used to explain data or provide hypotheses . DependiOne of the most detailed kinetic models of GSH metabolism was presented by Reed et al., . This moSeveral kinetic models have been developed in an effort to describe cancer metabolism with some including a number of reactions involving GSH. An interesting example of such a model published by Roy et al.,  consistsOne of the major advantages of detailed kinetic models is the possibility to include inhibition or activation regulation of reactions. Analysis of metabolic self-inhibition based on the enzymological data obtained through a century of experimentation results collected in metabolism databases such as BRENDA  has showSimulation of metabolism of GSH in the context of a complete metabolic network of cancer cells and tissues as well as metabolic interactions between cancer cells and their microenvironment is possible with either Boolean models or much more informative genome scale flux models . CurrentFinally, many tumor tissues include both cancer and healthy cells as well as possibly unique microorganism content within the microenvironment. Therefore, for computational models to provide true in silico laboratories and complete understanding of the role of GSH in this complicated system it will be necessary to simulate molecular single-cell effects in the context of a multicellular system. Current approaches for multi-cellular system modeling for cancer have been recently reviewed  and giveGSH exists in organelles, cells, tissues, extracellular vesicles, and body fluids in a reduced form or as a disulphide, in its oxidized form. The high reactivity of GSH makes exact measurement of absolute concentrations challenging, likely leading to many discrepancies in reported concentrations in various biological systems. Introduction of advanced analytical techniques including a variety of mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) approaches and platforms, as well as better methods for prevention of auto-oxidation now leads to more accurate measurement of the changes in GSH/GSSG concentrations. This is particularly important in the development and utilization of tumor therapies where GSH levels can be both an indicator of therapy success and a direct target to therapies. Development of intelligent and targeted tumor therapy that takes advantage of the very high demand of tumors for GSH requires further understanding of the many roles of this important molecule. More accurate measurement of GSH concentrations and its flux across cell organelles and between tumor cells and their microenvironment will allow development of better tools for in silico exploration of therapeutic opportunities and deeper understanding of glutathione’s many roles.It is also important to recognize that our knowledge of the role of GSH in tumor development, progression and therapy is based primarily on data from tumor or stromal cells grown in isolation under in vitro experimental conditions and caution should be exercised to extrapolate these findings to the complex in vivo TME. Attempts are being made to develop tumor organoid models with the possibility to provide better representation of redox processes in the tumor system and further work in this area is essential. Moreover, exosomes represent a heterogeneous population of vesicles and our inability to distinguish tumor from stromal cell-derived exosomes and their distinct cargoes add another layer of complexity. Therefore, it is necessary to develop a better toolkit and standardize methods of isolation and analysis of specific subset of exosomes from in vivo tumor tissue. Based on the recent studies that exosomes harbor an ‘off-the-shelf’ pool of metabolite cargo which can fuel tumorigenesis, preventing exosomes from providing nourishment to tumor cells might be an effective strategy for cancer treatment.Improvement in therapeutic targeting will require a better understanding and stronger focus on the TME and inflammation as an integral part of tumor progression with an integrated approach in the analysis of tumor and various stromal cells. Utilizing high-throughput measurement methods applied to the novel, more accurate and appropriate model systems will provide valuable data for the development of GSH metabolism and interaction models both as mechanistic, knowledge-based or machine-learning, data-based models providing platforms for drug target determination and investigation of complete effect of combined therapies. Major developments in the machine learning approaches for drug design including improvement in the drug and target parameterization methods are expected to provide novel, more specific and sensitive treatments and provide avenues for the development of safe multimodal cancer therapies. GSH, with its pleiotropic functions, deserves to have a prominent role in these developments.


Such responsiveness of BBB tight junctions to diseases indicates that these protein complexes are critical for maintenance of CNS homeostasis. In fulfillment of this vital role, BBB tight junctions are also a major obstacle to therapeutic drug delivery to the brain. There is an opportunity to overcome this substantial obstacle and optimize neuropharmacology via acquisition of a detailed understanding of BBB tight junction structure, function, and regulation. In this review, we discuss physiological characteristics of tight junction protein complexes and how these properties regulate delivery of therapeutics to the CNS for treatment of neurological diseases. Specifically, we will discuss modulation of tight junction structure, function, and regulation both in the context of disease states and in the setting of pharmacotherapy. In particular, we will highlight how these properties can be potentially manipulated at the molecular level to increase CNS drug levels via paracellular transport to the brain.The blood-brain barrier (BBB) allows the brain to selectively import nutrients and energy critical to neuronal function while simultaneously excluding neurotoxic substances from the peripheral circulation. In contrast to the highly permeable vasculature present in most organs that reside outside of the central nervous system (CNS), the BBB exhibits a high transendothelial electrical resistance (TEER) along with a low rate of transcytosis and greatly restricted paracellular permeability. The property of low paracellular permeability is controlled by tight junction (TJ) protein complexes that seal the paracellular route between apposing brain microvascular endothelial cells. Although tight junction protein complexes are principal contributors to physical barrier properties, they are not static in nature. Rather, tight junction protein complexes are highly dynamic structures, where expression and/or localization of individual constituent proteins can be modified in response to pathophysiological stressors. These stressors induce modifications to tight junction protein complexes that involve  The neurovascular unit (NVU) is an anatomical and functional unit that responds to the needs of neuronal energy demands by precisely regulating cerebral blood flow . The NVUin vivo transendothelial electrical resistance (TEER) of brain microvessels is 1,500–2,000 Ω cm2, which is considerably higher than peripheral microvessels that have a TEER of 3–30 Ω cm2  and two cytoplasmic domains that consist of a short amino (N)-terminal sequence and a long carboxyl (C)-terminal sequence. The ECLs of claudins contribute to the formation of the TJ backbones by homo‐ or hetero-dimerization or oligomerization with neighboring claudins on the same membrane (cis-interaction) and/or with those on the apposing cell  that have been identified at the mammalian BBB. These proteins are known as tricellulin, marvelD3, and occludin. Tricellulin plays a prominent role in regulating paracellular permeability to macromolecules  while main vitro experiments have repeatedly demonstrated that elevated phosphotyrosine and diminished phosphoserine/threonine are generally associated with disruptive barrier functions by interfering with occludin-ZO interactions and/or occludin trafficking , ZO-2 (160 kDa), and ZO-3 (130 kDa) belong to the PDZ domain-containing protein and membrane-associated granulated kinase (MAGUK) family, which constitute the most prominent intracellular proteins at the TJs. ZO family members are essential for assembly of functional TJs by binding to and cross-linking a wide range of TJ proteins and tethering them to the actin cytoskeleton . The ZO in vivo studies.BBB dysfunction is a pathological feature of several diseases affecting the CNS . BBB perAlzheimer’s disease (AD) is the most common dementia disorder and is associated with substantial neurovascular dysfunction and pathology . PatholoStudies in AD animal models display conflicting results regarding BBB permeability and TJ alterations. Tg2576 mice, which overexpress the amyloid precursor protein (APP) with the Swedish mutation (K670N and M671L) showed increased brain uptake of albumin and a fluorescein derivative following transcardial perfusion as well as increased extravasation of IgG and fibrin . Several2 activation, which leads to barrier leakage by activating the TJ degrading enzymes matrix metalloproteinase (MMP)-2 and MMP-9.A number of BBB impairments have been shown in epilepsy, which exhibits abnormal brain activity leading to seizures . IgG extHypoxia is a central component of cerebral ischemia, which is a restriction in blood supply to the CNS typically caused by a thrombus or embolism. Both hypoxia and ischemia have been shown to exhibit an increase in BBB permeability associated with TJ alterations . This in2) for 1 h], the permeability of the BBB increases to (14C)-sucrose (~340 Da). Of particular note, permeability to Evans blue pre-adsorbed to albumin (~80 kDa) was not altered, which suggests that acute hypoxia leads to BBB permeability changes to small molecules but not macromolecules -sucrose and degradation of claudin-5 and ZO-1 were inhibited by the broad spectrum MMP inhibitor  is a neurodegenerative disorder caused by an autosomal dominant mutation, which results in movement, cognitive, and psychiatric problems. Mutant Huntington protein has been shown to aggregate in the blood vessels of the caudate nucleus in HD patients and in brain tissue from R6/2 mice, a preclinical HD model . MRI stuMultiple sclerosis (MS) is a chronic, progressive CNS disorder, in which the immune system attacks myelinated fibers which causes inflammation, scar tissue, or lesions. BBB disruption during MS is thought to influence oligodendrocyte death, axonal damage, demyelination, and lesion development . Breakdoin vivo model of demyelinating diseases, have shown TJ alterations that correlate with changes in BBB permeability. Although imperfect, the EAE model does display some similarities in CNS pathologies with MS and, therefore, is commonly used as an animal model to study MS-associated mechanisms. Many studies conducted in experimental autoimmune encephalomyelitis (EAE), an in situ brain perfusion, we have shown in several models of peripheral inflammatory pain (PIP) that brain uptake increases to (14C)-sucrose, but not to Evans blue-albumin, suggesting that inflammatory pain alters BBB paracellular permeability to small molecules but not macromolecules -sucrose was observed beginning at 30 min with no changes in claudin-5 or occludin expression  is a neurodegenerative disorder, which affects movement. Evidence of BBB disruption in PD includes the findings of significant increase in albumin and IgG in the cerebrospinal fluid of PD patients  and an iTraumatic brain injury (TBI) is a complex, dynamic, and heterogenous pathology which can be classified as impact or non-impact, focal or diffuse, mild, moderate, or severe. A compromised BBB is a well-established pathological feature of TBI . Intracrin vivo.Although the BBB plays an important protective role of the CNS, modulation of TJs may allow increased brain delivery of drugs through the paracellular route . For exaOne strategy to increase brain delivery of drugs is intracarotid infusion of hyperosmotic concentrations of arabinose or mannitol. This technique causes reversible vasodilation and shrinkage of endothelial cells, which leads to a loosening of the TJs, an increase in paracellular diffusion, and bulk fluid flow across the BBB . While t2 receptor agonist RMP-7 (Cereport®) increased lanthanum penetration through TJs of the BBB by loosening the junctional complexes (G) from vibrio cholerae has been shown to increase BBB to (14C)-sucrose, likely by activating PKC  bursts generated from an external source are able to utilize microbubbles as contrast agents that act as cavitation sites, which physically disrupt the BBB . IncreasTJ protein complexes are the major determinants of paracellular permeability at the BBB. While brain microvascular endothelial TJs function to protect the CNS from potentially harmful substances in the blood, their protective role also represents a major obstacle to effectively deliver therapeutics for treatment of neurological diseases. Evidence is increasingly emerging that pathological alterations of TJs at the BBB may exacerbate or possibly initiate neurological dysfunction. A better understanding of how TJ expression, localization, and function is altered at the BBB during disease states may provide opportunities to protect the CNS from adverse consequences resulting from BBB disruption. Conversely, understanding the mechanisms involved in rapidly and transiently regulating TJ function at the BBB may allow improved delivery of therapeutics to the CNS through the paracellular route. Future research elucidating signaling pathways affecting TJ function at the BBB has the potential to both protect the CNS from a compromised barrier and more effectively treat neurological disorders by optimizing drug delivery.JL and JY wrote the paper. PR and JL created the figures and tables. PR and TD edited the paper. 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.


Ultraviolet (UV)-induced cationic frontal polymerization has emerged as a novel technique that allows rapid curing of various epoxy monomers upon UV irradiation within a few seconds. In the presence of a diaryliodonium salt photoinitiator together with a thermal radical initiator, the cationic ring opening polymerization of an epoxide monomer is auto-accelerated in the form of a self-propagating front upon UV irradiation. This hot propagating front generates the required enthalpy to sustain curing reaction throughout the resin formulation without further need for UV irradiation. This unique reaction pathway makes the cationic frontal polymerization a promising route towards the efficient curing of epoxy-based thermosetting resins and related composite structures. This review represents a comprehensive overview of the mechanism and progress of UV-induced cationic frontal polymerization of epoxy monomers that have been reported so far in literature. At the same time, this review covers important aspects on the frontal polymerization of various epoxide monomers involving the chemistry of the initiators, the effect of appropriate sensitizers, diluents and fillers. In the past few decades, polymer resins cured via frontal polymerization technique have been the focus of research for applications such as nanocomposites, functionally graded polymers, sensory materials, hydrogels and fibre-reinforced composites ,4,5,6,7.In particular, ultraviolet (UV)-induced frontal polymerization has attracted a great number of industrial sectors for various applications due to its low operational costs, ambient temperature processing, solvent-less formulation and the freedom to have tuneable final properties of the cured polymer . UV-curiWhile photoinduced radical chain growth polymerization has been widely investigated and exploited from an industrial point of view, it was not until the discovery of onium salt photoinitiators by James V. Crivello in the 1970s that the first cationic photoinduced polymerization was reported . TypicalHowever, the main limitation of UV-induced crosslinking reaction is related to the low penetration depth of UV light, which limits polymerization techniques, even cationic photopolymerization, to thin films and adhesive applications ,32. The In 2004, Mariani et al.  proposedThe purpose of the current review is to give a comprehensive overview of studies on RICFP that have been investigated in the literature so far. Since epoxy-based monomers are the most widely used components for transport and construction sectors, this review is also aimed for providing a guide for UV frontal curing of various epoxy monomers used commercially. A few authors have presented reviews on UV-activated cationic polymerization ,43,44, bIn the past, organometallic-based photoinitiators have been developed and employed for cationic photopolymerization of epoxides. Some of the photoinitiators included manganese decacarbonyl , complex−, Cl−, Br− generate weak Brønsted acids and the ion pairs collapse to give stable covalently bonded compounds. The second type of anions, including HSO4−, ClO4− etc., are called intermediate nucleophilic anions, while SbF6−, AsF6−, PF6−, BF4− are termed as weakly nucleophilic anions phenyl] phenyliodonium hexafluoroantimonate (λmax = 250 nm)  benzene (BOB) and compared with their respective SbF6 and (BC6F5)4− anions. It was found that both diphenyliodonium and triarylsulfonium salts based on Al anion exhibited higher polymerization rates and monomer conversion than their corresponding SbF6- and (BC6F5)4− anions -1,8-naphthalimide ..41].With this review, we provide a useful toolbox for the UV-triggered cationic frontal polymerization of various epoxide monomers that employ an iodonium salt photoinitiator and a thermal radical initiator. Key aspects influencing the polymerization kinetics and related thermo-mechanical properties are summarized in a comprehensive way. In particular, an overview of the chemistry of selected iodonium salts and thermal initiators is given, while studies on the cationic photopolymerization of various epoxide monomers are discussed and arranged chronologically. In addition, the use of alternative light sources for initiating the cationic polymerization is highlighted together with the effect of sensitizers on cure kinetics and front parameters. The structure–reactivity relationship and effect of reactive diluents on the polymerization of various epoxide monomers is detailed. In the last section, the transfer of the UV-triggered cationic frontal polymerization from neat resins to polymer-based composites is discussed. Particular focus is on the effect of various fillers and fibres on the radical-induced cationic frontal polymerization of epoxy-based resin formulations.2 corrugated carbon fibre-reinforced polymer composite (FRPC) panel in only 5 min and by spending 750 J of energy. From the results of a study on the energy consumed versus time required to cure composites, the group calculated that the curing of a section of the Boeing 787 fuselage by FROMP reduces the energy consumption by ten orders of magnitude compared with autoclave processing technique [Cationic frontal polymerization of epoxide monomers has opened up a new path towards a rapid, low cost and energy-independent curing of epoxy-based resins. Recent studies by Knaack et al. , predictechnique .In the production of composite materials, the product cycle time, total energy consumption, fibre–matrix adhesion and the quality of the cured composite are crucial parameters that are presently a subject of improvement via an intensive researching. Particularly for thermosetting resins such as epoxide monomers, maximum monomer conversion or high cross-link density and uniform distribution within the fibre/filler can be improved by employing frontal polymerization. This technique does not only improve the final product quality but also cuts down a lot in terms of input energy requirements for thermal curing and subsequent post curing, as well as eliminating the usage of toxic solvents for dissolution of the curing agents. Furthermore, complex part geometries that are either not fully accessible during a conventional curing process can now be easily accessed via frontal polymerization at ambient processing temperature and without the necessity of tools for curing under high-pressure.The kinetics of frontal polymerization allows the use of various other monomers that are particularly used to improve the mechanical properties of neat epoxy resins. This means that any functionalized monomer that do not act as radical scavengers during the initial photo cleavage of the iodonium salt and also has appreciable polarity to solubilize with epoxy monomers can be used to prepare epoxy blends of desired thermomechanical properties. Epoxidized elastomers are an interesting class of monomers that can be employed also in the fabrication of high tensile and impact strength composites, which were firstly reported by Crivello and Yang in 1994 . Due to Frontal polymerization was also acknowledged by Pojman et al.  as a potWith recent successful studies focused on the thermomechanical properties of fibre/filler-reinforced epoxy composites ,42,125,


To the Editor,Large scale multi-omics analysis has identified significant differences in the biomarkers between COVID-19 disease and control subjects  and PCOS146 PCOS and 97 control women who presented sequentially to the Department of Endocrinology, Hull and East Yorkshire Hospitals NHS Trust were recruited to the local PCOS biobank (ISRCTN70196169) . PCOS diAs reported previously , cohortsFor the 60 protein biomarkers described previously, 21 were found to differ in PCOS: for vessel damage 7 of 16 proteins differed, for platelet degranulation 2 of 11 proteins differed, for the coagulation cascade 9 of 24 proteins differed and for the acute phase response 3 of 9 proteins differed (Table These data show that altered protein expression relative to controls may occur in other conditions such as PCOS, and that COVID-19 biomarker changes found between respiratory patients with and without COVID-19 require validation before they can be confirmed to be related to COVID-19 disease and its severity. It is of concern that a number of significant protein biomarkers described for COVID-19 patients and its severity were also found in PCOS, spanning the biological processes involved in vessel damage, platelet degranulation, the coagulation cascade and the acute phase response, perhaps indicating why these patients may be at risk of more severe COVID-19 disease .Limitations of the study include utilizing a different method of proteomic analysis compared to others that may not be directly comparable .In conclusion, 21 of 60 protein biomarkers reported in respiratory patients with COVID-19 were found to differ between women with and without PCOS, showing the necessity for validation of such biomarkers, and suggesting that more severe COVID-19 disease may occur in PCOS.


Cholestatic liver diseases are a significant cause of morbidity and mortality and the leading indication for pediatric liver transplant. These include diseases such as biliary atresia, Alagille syndrome, progressive intrahepatic cholestasis entities, ductal plate abnormalities including Caroli syndrome and congenital hepatic fibrosis, primary sclerosing cholangitis, bile acid synthesis defects, and certain metabolic disease. Medical management of these patients typically includes supportive care for complications of chronic cholestasis including malnutrition, pruritus, and portal hypertension. However, there are limited effective interventions to prevent progressive liver damage in these diseases, leaving clinicians to ultimately rely on liver transplantation in many cases. Agents such as ursodeoxycholic acid, bile acid sequestrants, and rifampicin have been mainstays of treatment for years with the understanding that they may decrease or alter the composition of the bile acid pool, though clinical response to these medications is frequently insufficient and their effects on disease progression remain limited. Recently, animal and human studies have identified potential new therapeutic targets which may disrupt the enterohepatic circulation of bile acids, alter the expression of bile acid transporters or decrease the production of bile acids. In this article, we will review bile formation, bile acid signaling, and the relevance for current and newer therapies for pediatric cholestasis. We will also highlight further areas of potential targets for medical intervention for pediatric cholestatic liver diseases. Causes of chronic liver injury in children include a wide variety of congenital and acquired diseases. The primary cholestatic diseases of infancy and childhood are frequently symptomatic and often rapidly progressive. These chronic cholestatic diseases that present in childhood include biliary atresia (the most common cause of cholestatic liver disease in children), Alagille syndrome, progressive familial intrahepatic cholestasis diseases (PFIC), bile acid synthesis defects, cystic fibrosis related liver disease, ductal plate abnormalities including Caroli syndrome and congenital hepatic fibrosis, primary sclerosing cholangitis (PSC), and certain metabolic diseases.Many patients ultimately require liver transplantation and within the pediatric population, cholestatic liver diseases remains the leading indication for pediatric liver transplant in the U.S, accounting for nearly half of all pediatric patients listed for liver transplant . For thiin vivo vs. in vitro) and the species incurring the injury; and these detailed mechanisms of cholestatic injury have been reviewed in detail elsewhere  which often leads to clinical symptoms and serum laboratory abnormalities. The production of bile is influenced greatly by the enterohepatic circulation and recycling of bile acids. The impediment to bile flow in cholestasis may be due to obstructive causes  or secondary to any impairment of appropriate synthesis or secretion of the components of bile from the hepatocytes and cholangiocytes. The formation of bile and its flow into the intestinal lumen serves both as an excretory function of the liver within its role to metabolize and detoxify substances, and as an aid in digestion of fat and fat-soluble vitamins. When bile acids are retained within cells, their detergent nature and induced signaling pathways can lead to significant cell damage. The exact mechanism of injury in human and animal models of cholestasis is likely multifactorial  and may be different depending on the precise nature of the injury, the setting of injury  and chenodeoxycholic acid (CDCA) in humans by a complex biochemical pathway involving a number of different hepatic enzymes within the “neutral” (classic) pathway or the “acidic” bile acid synthesis pathways. In the initial and rate limiting step within the neutral pathway, cholesterol is modified with the addition of a hydroxyl group at the C-7 position by the microsomal cytochrome P450 liver-specific enzyme cholesterol 7α-hydroxylase (CYP7A1) , 4. In tAfter primary bile acids are produced, they are conjugated to either glycine or taurine largely within peroxisomes . SeveralABCB11. Mutations in ABCB11 lead to the disease PFIC type 2 in humans  which is encoded by the gene n humans . After bn humans –20.ABCB4) which is known as mdr2 in mice  and intrahepatic cholestasis of pregnancy (ICP) (ATP8B1 gene) which is located at the hepatocyte canalicular membrane, and apical membrane of cholangiocytes and enterocytes , a nuclear receptor critical to bile acid homeostasis (TJP2), also known as zona-occludens 2, can lead to progressive intrahepatic cholestasis and has been referred to as PFIC4, however as more newly discovered causes of inherited progressive cholestasis are discovered, naming of the intrahepatic cholestasis diseases based on the mutated gene rather than a numbering system initially developed at a time prior to identification of the responsible mutations is superior.In addition to BSEP, there are several other specific transporters at the canalicular membrane that are responsible for excreting the other components of bile across this membrane. Phospholipids, primarily phosphatidylcholine (PC), are secreted via the multidrug resistance P-glycoprotein 3 in humans  , 24. PFIerocytes . The exaeostasis , 27. AddABCG5/ABCG8) also called sterolin, and mutations in ABCG5/ABCG8 genes can cause sitosterolemia, which has a varied clinical presentation including associated liver disease (ABCC2), and mutations in ABCC2 lead to Dubin-Johnson syndrome  located on the hepatocyte sinusoidal membrane which serve to reabsorb conjugated bilirubin , a chloride channel important for bicarbonate secretion into bile, is located on the apical membrane of cholangiocytes, and the chloride/bicarbonate exchanger AE2 (gene SLC4A2) is on the apical membrane of cholangiocytes and on the canalicular membrane surface (CFTR lead to cystic fibrosis and cystic fibrosis related liver disease (CFRLD). AE2 knock-out mice develop a phenotype similar to the adult cholestatic liver disease primary biliary cholanigitis (PBC)  . Bile flis (PBC) .Jagged1 (JAG1) or Notch2. Ductal plate malformations refer to cholangiopathies associated with the lack of normal development and remodeling of the intrahepatic bile ducts that occurs along the branches of the developing portal vein. These include entities such as Caroli syndrome and congenital hepatic fibrosis which are most commonly associated with autosomal recessive polycystic kidney disease (ARPKD), secondary to mutations in the gene PKHD1 which encodes fibrocystin  and lithocholic acid (LCA), respectively, which constitute the majority of bile acids excreted into the feces in humans , 36, 37.SLC10A2 (ABCC3) and MRP4 (encoded by gene ABCC4), in addition to OSTα-OSTβ as mentioned above  also known as the ileal bile acid transporter (IBAT) encoded by the gene SLC10A2 , 44. ASBSLC10A2 . ASBT trSLC10A2 . Bile acSLC10A2 , 46. At SLC10A2 , 48. TheSLC10A2 . During ed above . A in deed above .NR1H4 gene—nuclear receptor subfamily 1, group H, member 4) is a nuclear hormone receptor that influences bile acid synthesis by forming a heterodimer with retinoid X receptor (RXR) and binding directly to the regulatory elements  of its target genes, as well as by regulating the downstream transcription factor small heterodimer partner (SHP)  that are activated by bile acids and that have significant effects on the regulation of bile acid synthesis and metabolism. Most notably, the discovery of the first BAR, farnesoid X receptor (FXR), has led to dramatic reshaping of the understanding of the tight regulation of this process. FXR (encoded by the er (SHP) –54. Mutautations .After activation by bile acids, FXR regulates several aspects of bile acid trafficking and production. FXR induces the expression of SHP which binds another receptor, LRH-1 (liver receptor homolog 1), preventing it from interacting with promotor regions on both CYP7A1 and CYP8B1, therefore negatively regulating bile acid synthesis within the hepatocyte . FXR alsABCB11 , the pregnenolone X receptor , the vitamin D receptor (VDR), and the constitutive androstane receptor (CAR). PXR is activated by LCA and likely serves to reduce bile acid toxicity via its regulation of CYP7a1 and other cytochrome p450 enzymes, OATP2, and MRP2 –66.An alternative family of receptors known as the G-protein-coupled receptor family, the most notable of which is GPBAR1 also known as TGR5 , are also able to bind bile acids  –69. TGR5The structure and conjugation of each bile acid affects its hydrophobicity which influences the ability to activate the different receptors discussed above. CDCA is the most potent naturally occurring ligand of FXR followed in strength by DCA = LCA > CA , 73, 74.in vitro data has shown that the UPR pathways function to reduce ER stress and hepatic injury as well as regulate bile acid synthesis and transport, and that activation of FXR in turn also influences the UPR  is an adaptive cellular response pathway to endoplasmic reticulum (ER) stress that functions to regulate protein homeostasis, but can also trigger apoptosis. Activation of the UPR has been described in several liver diseases including fatty liver disease, viral hepatitis, and cholestasis , 76. How the UPR . ER stre the UPR , 79.Historically, pediatric chronic cholestasis due to all causes has primarily been managed symptomatically. Pediatric patients with cholestatic liver diseases frequently suffer from significant symptoms and complications including poor growth secondary to fat malabsorption, fat soluble vitamin deficiencies, hepatic osteodystrophy, and complications related to progressive portal hypertension such as ascites and gastrointestinal bleeding. Significant chronic pruritus is common in several of the genetic syndromes and a significant cause of major morbidity. Agents such as ursodeoxycholic acid, bile acid sequestrants, and rifampicin have been the mainstays of treatment for years with the understanding that they may promote choleresis or alter the composition of the bile acid pool. However, patient response to these medications is frequently insufficient and data to show that they alter disease progression in most conditions remains lacking. Patients frequently require high calorie supplemental formulas to improve malnutrition secondary to fat malabsorption and antihistamines and other medications to aid in pruritus management and sleep. Fat soluble vitamin levels should be monitored closely and repleted if required, though deficiencies may be difficult to correct , 81. Howin vitro and in rodent models and the full range of effects in human disease are still uncertain. In adults with PSC, there were several studies that showed improvement in biochemical markers on UDCA (on doses between 10 and 15 mg/kg/day), but failed to show improvement in long term outcomes  and has been shown to improve long term outcomes including reduction in the risk of death or need for liver transplant for PBC patients . Howeveroutcomes –96. Addioutcomes . The curoutcomes . Howeveroutcomes –102. A routcomes . This suoutcomes . A recenoutcomes .Despite this lack of strong evidence in favor of its use, given the low side effect risk profile of standard dose UDCA (10–20 mg/kg/day), it is often employed in cases of pediatric chronic cholestasis.−/−) mouse model of PSC and PFIC3, NorUDCA has been shown to be more effective than UDCA in preventing liver disease progression, and there is some evidence that NorUDCA may have anti-fibrotic effects  are a group of rare metabolic diseases that are characterized by single enzyme defects (SED) in bile acid synthesis pathway proteins or Zellweger spectrum disorders (ZSDs). Oral cholic acid was approved for use in BASD in 2015 and has been shown to decrease synthesis of toxic bile acid intermediates, and improve histologic features on liver biopsy in certain patients with BASD , 113. Thin vitro data), these studies have also shown a decrease in secondary, hydrophobic bile acids (LCA and DCA). Therefore, alternative explanations for a reduction in pruritus may include alterations of intestinal flora which alters production of secondary bile acids, or additional excretion of other possibly pruritogenic compounds via increased expression of MRP2 or other liver canalicular membrane transporters  through several proposed mechanisms including increased hydrophilic hepatic/biliary bile acids, and increasing colonic resin bound BA which are more abundant TGR5 ligands, which raises the possibility of specific benefits of bile acid sequestrants in PSC and PFIC3 outside of simply treating pruritus , has been used for treatment of hyperammonemia secondary to urea cycle defects given its nitrogen scavenging properties. However, it is also a chemical chaperone that may bind to areas of misfolded protein, preventing aggregation, enhance proper protein folding, and therefore increasing delivery of such proteins to their target locations . 4-PBA h−/− mice , now commonly referred to as obeticholic acid (OCA), was developed and has an FXR agonist potency that is 100-fold greater than CDCA , 141. OC−/−) mouse model of PSC and PFIC3, INT-767 treatment lead to improved biliary fibrosis and hepatic inflammation as well as induced bicarbonate rich bile production which was superior to improvements seen in selective FXR and TGR5 (GPBAR1) treatments alone  has been developed that is reported to be a 3-fold more potent FXR ligand than OCA and is also a TGR5 (GPBAR1) ligand. In the mdr2 are ongoing in adults with PBC, PSC, and NASH and preliminary human trials indicate that they may have potential therapeutic benefits with less side effects including pruritus –148. GivFXR activation promotes the release of ileal FGF19 (the human homolog of murine FGF15) which suppresses bile acid synthesis by repressing CYP7A1 expression and therefore FGF19 analogs may be a potential therapeutic target for pediatric cholestatic liver diseases. However, there are concerns that FGF19 over-expression in mice lead to hepatocellular carcinoma, and concerns about a potential risk of the development of cholangiocarcinoma in cholestatic biliary diseases . HoweverGiven the often rapidly progressive nature of fibrosis and fibro-inflammatory liver damage in many pediatric cholestatic diseases, agents that specifically reduce inflammation and hepatic fibrosis are desirable. Unfortunately, the limited number of clinical trials that have attempted to modulate fibro-inflammatory responses in biliary atresia, specifically with corticosteroids and intravenous immunoglobulin, have not demonstrated any improvement in outcomes , 153. In−/− mice with MDR3-expressing hepatocytes while on a standard diet lead to modest improvement in phospholipid excretion in mice, but did lead to histologic improvement of disease and appeared to decrease the development of hepatic tumors   . Prior wdilator) . Howeverdilator) . This stdilator) . A follodilator) . Though dilator) , 161.Stem cell infusions for patients with liver disease has been proposed to alter hepatic inflammation and fibrosis which makes it a possible favorable treatment for pediatric cholestatic liver diseases. Additionally, if autologous stem cells are used then there would not be a requirement for immunosuppression (unlike hepatocyte transplantation). In a mouse model of biliary atresia, animals were given bone marrow-derived mesenchymal stem cells via intraperitoneal injection and the authors reported significantly improved AST, ALT, total and direct bilirubin along with improved histologic features and markers of hepatic fibrosis 14 days later . There iGiven the prevalence of diseases with known, single-gene mutations within the field of chronic pediatric cholestasis, gene-editing technologies are an attractive future therapeutic option. There have been human subjects treated with CRISPR edited T cells and hematopoietic stem and progenitor cells and clinical trials are ongoing . While there are no current studies underway with pediatric cholestasis, CRISPR/Cas-9 gene-editing or and other prime editing tools may offer promise for future gene editing therapies for patients with single gene or other known mutations.Chronic cholestatic liver diseases are a significant cause of morbidity and mortality within the pediatric population and there is a notable lack of specific medical therapies that improve outcomes in these patients. However, the past few decades a new understanding of the intricate signaling pathways involved in bile acid metabolism and transport has led to novel targets for treating these diseases in pediatric patients. These include strategies to limit cytotoxicity by changing the bile pool hydrophobicity, enhancing protein folding, altering the expression of bile acid and other liver and ileal transporters to promote choleresis, enhancing hepatic detoxifying enzymes, disrupting the enterohepatic circulation of bile acids and decreasing the production of bile acids. Other strategies can target signaling pathways to decrease inflammation and fibrosis in liver diseases, and potential future gene editing therapies may be promising for identified gene defects. Based on our ever increasing and more nuanced understanding of the underlying etiologies of specific patient genotypes and phenotypes with pediatric cholestasis, combination therapies tailored to specific patients may be employed in the future. However, additional studies are warranted to elucidate potential therapeutic agents in humans that are effective, and also have favorable tolerability and low risk of long-term side effects.AK and RG contributed to this manuscript, to the concept of the work, to the drafting and revisions of the manuscript, gave final approval of the manuscript, and are in agreement to be accountable for all aspects of the work.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.


Panax notoginseng saponins) in preventing deep venous thrombosis (DVT) of lower extremity after orthopedic surgery.  To systematically evaluate the clinical efficacy of Xueshuantong injection  of Xueshuantong injection in prevention of lower extremity DVT after orthopedic surgery were retrieved from CNKI, Wanfang database, VIP, PubMed, and Cochrane Library by August 2020. Revman5.2 was used to analyze the results.  A total of 20 articles including 2336 patients were included. The results of meta-analysis showed that the incidence of DVT in the experimental group was lower than that in the control group; after operation, the D-dimer (Ddimer), thrombin time (APTT), and prothrombin time (PT) in the experimental group were significantly improved compared with those in the control group, and the difference between the two groups was statistically significant.  Xueshuantong injection can effectively prevent the formation of lower extremity DVT after orthopedic surgery and antagonize the postoperative hypercoagulable state of blood, which has high clinical value. Panax notoginseng saponins) has antiplatelet aggregation effect and is widely used in orthopedic surgery for thromboprophylaxis , I2 = 99%, p < 0.00001); therefore, a random effect model was performed for a meta-analysis, which showed that Xueshuantong injection or it combined with other treatment can reduce patients' D-D level ]; 10 studies measured D-D levels in patients after fracture surgery. There was a statistically significant degree of heterogeneity among individual studies ; therefore, a random effect model was performed for a meta-analysis, which showed that Xueshuantong injection or it combined with other treatment can improve patients' PT level ]; Nine studies observed PT levels in patients after fracture surgery. There was a statistically significant degree of heterogeneity among individual studies ; therefore, a random effect model was performed for a meta-analysis, which showed that Xueshuantong injection or it combined with other treatment can improve patients' APTT level ]; A total of 9 articles measured APTT. There was a statistically significant degree of heterogeneity among individual studies  Although all the included literatures mentioned the method of randomized grouping, some of them lacked a detailed description of the concealment of the randomized scheme; (2) the number of included literatures was small, all of them were Chinese literatures, and there were some problems such as large sample size gap between literatures, insufficient detailed basic data, and incomplete unification of treatment cycle; (3) all the included literatures were published articles. If there are no unpublished results, studies with negative results may be missed, and there is a risk of reducing the strength of the argument. Therefore, the evidence strength of the conclusion of this study needs to be further improved, and more high-quality RCTs are needed to verify, so as to obtain a more accurate conclusion on the effect of Xueshuantong injection in preventing lower extremity deep vein thrombosis after fracture surgery, so as to provide good clinical guidance for the prevention of lower extremity deep vein thrombosis after fracture surgery.


EAAC1) is a type of neuronal glutamate transporter and considered to have an additional action involving the neuronal uptake of cysteine, which acts as a crucial substrate for glutathione synthesis. Previously, our lab demonstrated that genetic deletion of EAAC1 leads to decreased neuronal glutathione synthesis, increased oxidative stress, and subsequent cognitive impairment. Therefore, we hypothesized that reduced neuronal transport of cysteine due to deletion of the EAAC1 gene might exacerbate neuronal injury and impair adult neurogenesis in the hippocampus after transient cerebral ischemia. EAAC1 gene deletion profoundly increased ischemia-induced neuronal death by decreasing the antioxidant capacity. In addition, genetic deletion of EAAC1 also decreased the overall neurogenesis processes, such as cell proliferation, differentiation, and survival, after cerebral ischemia. These studies strongly support our hypothesis that EAAC1 is crucial for the survival of newly generated neurons, as well as mature neurons, in both physiological and pathological conditions. Here, we present a comprehensive review of the role of EAAC1 in neuronal death and neurogenesis induced by ischemic stroke, focusing on its potential cellular and molecular mechanisms.Although there have been substantial advances in knowledge regarding the mechanisms of neuron death after stroke, effective therapeutic measures for stroke are still insufficient. Excitatory amino acid carrier 1 ( EAAC1), and it carries out this function by increasing the cysteine concentration in the cell. EAAC1, also known as SLC1A1 or excitatory amino acid transporter 3 (EAAT3), increases intracellular glutathione (GSH) levels by importing cysteine from the extracellular space. The process includes the expression of the gene that affects neurons -thymidine. These cells can also be marked by retrovirus-related substances that detect the M-phase of neurons, such as green fluorescent protein (GFP) or LacZ. Other substances, such as Ki67, collapsin response mediator protein 4 (CRMP4) , polysialic acid–neural cell adhesion molecule (PSA–NCAM), SOX gene families, and calretinin, are used to detect dividing neurons. Neurons that are differentiating can be detected by doublecortin (DCX), proopiomelanocortin (POMC), or neuronal nuclei (NeuN) antibodies. It has been reported that newly generated neurons in the dentate gyrus and striatum are GABAergic interneurons, with the former requiring two maturation steps and the latter needing only one ,43,45,46Although stem cells in the CNS are in comparatively low oxidative conditions compared to neurons in other organs , they ar2+ circumstances, it is clear that this imbalance impairs cellular division in the DG [Because the reperfusion state causes the unregulated elevation of the oxidation level in low-pH and high-Can the DG . Inflictn the DG . As a ren the DG ,46,51.EAAC1 as the main cysteine importer of cysteine in hippocampal regional mitosis. Our previous study showed that EAAC1 is important for the survival of newborn neurons in the adult hippocampus under physiological and ischemic conditions [EAAC1−/− mice under ischemia-free conditions compared to the age-matched WT mice. However, EAAC1−/− mice showed poor survival outcomes of newly generated neurons in both young and aged mice. In addition, EAAC1−/− mice had fewer progenitor cells and their subsequent neuroblasts 7 days after ischemia. The neuronal maturation and survival rate of newly generated cells were also decreased in EAAC1−/− mice 30 days after ischemia  is not essential for novel proliferation in the DG, which illuminates the importance of nditions . We founischemia . This clIn the sites where the pathological nucleic acid and lipids were analyzed, oxidative stress can be a problem in neurogenesis in the aspect of blocking proliferation of normal type I cells. Studies show decreased efficiency of pyruvate dehydrogenase and other types of mitochondrial dysfunction during the first 30–45 min of reperfusion, during which the number of hydroxyl radicals is at its maximum ; the memEAAC1 is one of the important neuroprotectants in the aspect of oxidative stress, manipulating factors related to EAAC1 will multiply the effect of the gene on neural death and neurogenesis. The main interference involves factors that reinforce the expression of EAAC1 in the cell membrane or lessen factors that impair the expression of EAAC1. As the expression of EAAC1 is inhibited by GTRAP3-18, inhibition of GTRAP3-18 results in an increase in EAAC1. Many studies have revealed that protein expression can be induced by adding MeβCD, altering the cellular GSH level. This may be rooted in MeβCD elevating cAMP response element (CRE)-mediated gene expression and extracellular signal-related kinase (ERK) as cholesterol is cellularly discharged, both promoting the expression of GTRAP3-18 and physically impeding EAAC1 [EAAC1 is done through the same pathway, finding that a cholesterol-rich glial culture medium increases glutamate transport means that the existence of extracellular cholesterol impedes the synthesis of GSH by changing the membrane composition of neural cells [EAAC1 are shown by the accumulation of metals such as Mn. A distinct negative correspondence between injected MnCl2 and mRNAs of the transporter, and the same variable with the amount of xCT-positive astrocytes, was detected in the striatum of the brain in neonatal mice. The phenomenon involves alteration of EAAC1 and xCT directly by the metal ion itself, altering the cysteine level in the microcellular environment and increasing oxidative stress [EAAC1.As ng EAAC1 ,55. As tal cells . Some ste stress . TherefoEAAC1′s role in producing GSH in neurons, the addition of a substance that can promote the cellular production of GSH itself will reduce cell death and increase functional neurons during the reperfusion state. Such an example is NAC. The effectiveness of NAC has been shown in many studies on the ischemia–reperfusion state to prove that defects of EAAC1 are related to neural damage and neural production at the same time because NAC provides cellular cysteine [Considering cysteine ,46. The Considering increases in mitotic factors such as basic fibroblast growth factor (bFGF) and inflammatory process inducers such as nitric oxide synthase, leukotrienes may promote a cellular deficit from ischemia . In the EAAC1 and both neuronal death and neurogenesis, it is evident that the oxidative stress from an outside stimulus is a critical factor in these phenomena. EAAC1 was proved to have more significant functions in cysteine uptake for the synthesis of GSH, which, in turn, controls the oxidative level of cells. This was more evident in ischemia models. After an ischemic event, an increased level of GSH helped lessen the number of degenerating cells. The main mechanism is the diminishing of oxidative stress from the outside stimulus and saving neurons from the crisis. For neuronal plasticity, a controlled oxidative level increased the number of cells that correctly matured, and GSH seems to support the differentiation of new cells, therefore increasing the rate of real neurogenesis and the formation of functional cell layers.Through deep insights about the relationship between EAAC1 expression in neurons would bring about the same neuroprotective effects. Achieving this result would fortify current theories indicating a linkage between EAAC1 and oxidative stress. For example, more studies should be performed on the relationship between GTRAP3-18 and cellular death or cellular regeneration. Understanding how it is linked to cytoskeletal changes during neuronal development gives insights into this aspect. Substances that are linked to fetal neural development, such as retinoic acids, need more investigation to uncover the role of oxidative stress in cellular processes that include not only correct maturation but also cellular development itself.Further studies should focus on whether a direct increase in


We review here the evidence for participation of mitochondrial autoimmunity in BC inception and progression and propose a new paradigm that may challenge the prevailing thinking in oncogenesis by suggesting that mitochondrial autoimmunity is a major contributor to breast carcinogenesis and probably to the inception and progression of other solid tumors. It has been shown that MNRR1 mediated mitochondrial-nuclear function promotes BC cell growth and migration and the development of metastasis and constitutes a proof of concept supporting the participation of mitochondrial autoimmunity in breast carcinogenesis. The resemblance of the autoantibody profile in BC detected by IFA with that in the rheumatic autoimmune diseases suggested that studies on the autoantibody response to tumor associated antigens and the characterization of the mtDNA- and nDNA-encoded antigens may provide functional data on breast carcinogenesis. We also review the studies supporting the view that a panel of autoreactive nDNA-encoded mitochondrial antigens in addition to MNRR1 may be involved in breast carcinogenesis. These include GAPDH, PKM2, GSTP1, SPATA5, MFF, ncRNA PINK1-AS/DDOST as probably contributing to BC progression and metastases and the evidence suggesting that DDX21 orchestrates a complex signaling network with participation of JUND and ATF3 driving chronic inflammation and breast tumorigenesis. We suggest that the widespread autoreactivity of mtDNA- and nDNA-encoded mitochondrial proteins found in BC sera may be the reflection of autoimmunity triggered by mitochondrial and non-mitochondrial tumor associated antigens involved in multiple tumorigenic pathways. Furthermore, we suggest that mitochondrial proteins may contribute to mitochondrial dysfunction in BC even if mitochondrial respiration is found to be within normal limits. However, although the studies show that mitochondrial autoimmunity is a major factor in breast cancer inception and progression, it is not the only factor since there is a multiplex autoantibody profile targeting centrosome and stem cell antigens as well as anti-idiotypic antibodies, revealing the complex signaling network involved in breast carcinogenesis. In summary, the studies reviewed here open new, unexpected therapeutic avenues for cancer prevention and treatment of patients with cancer derived from an entirely new perspective of breast carcinogenesis. Recently, Aras et al. reported that MNRR1, a nuclear DNA (nDNA)-encoded mitochondrial antigen, promotes cancer cell migration and the development of metastasis as a proof of concept supporting the participation of mitochondrial autoimmunity in breast carcinogenesis . This study also suggests that a panel of mitochondrial and non-mitochondrial antibodies has screening potential for breast and possibly other malignancies. These suggestions must be confirmed and deserve further investigation.The resemblance of the autoantibody profile in BC with that in the RADs such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) by IFA  suggesteThe autoimmune nature of the molecular changes observed in PBC and in PV ,12, the The basic approach used for biomarker discovery – to isolate and characterize the putative TAAs, including the construction of cDNA expression libraries, immunoscreening of cDNA libraries of potential BC autoantigens, the assembly of micro-collections of the cloned phages on derivatized glass slides, hybridization of the cloned phages with sera from cases and non-cancer sera, development of the autoantigen microarray, identification of unique clones using PCR, cDNA sequence determination, and homology searches of informative phages in databases – have all been reported –17 and wSera containing autoantibodies of unknown specificity had been used successfully in previous works for immune screening the cDNA library of potential BC autoantigens. However, in experiments aimed to identify BC biomarkers, the use of biopanning BC sera containing an abundant unknown 56 kDa antigen led to the identification of Annexin XIA as a prominent BC autoantigen . Based oThe reports suggesting that epithelial cancer cells participate in immunoglobulin synthesis –22 prompSince commercially obtained libraries are usually derived from a single malignant tumor, and given the heterogeneity of BC , a multic oxidase subunit MT-CO1 . Zhang et al. reported that DDX21 is highly expressed in BC tissue and in established cell lines, its protein expression levels correlate with cell proliferation rate, and it is induced by EGF signaling [Fourteen clones of the DEAD box protein DDX21 were identified in the RP cDNA library that were significantly associated with BC . DDX21 iignaling .Activated PARP-1 ADP-ribosylates DDX21 and promotes rDNA transcription . ElevateATF3 gene encodes a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB). This stress-responsive, cyclic AMP-dependent transcription factor is also involved in tumorigenesis. Relevant to the possible effect of autoreactive ATF3 in BC, this transcription factor interacts with ERK/MAPK signaling and with pathways involved in glucose/energy metabolism that are involved in carcinogenesis [ATF3, an adaptive-response gene, enhances TGFβ signaling and cancer-initiating cell features in BC cells. The study of Zhang et al. also reported that elevated DDX21 regulates c-JUN activity and rRNA processing in human BC [It is revealing that AP-1 itself is also targeted by autoantibodies in BC since the JUND Proto-Oncogene AP-1 Transcription Factor Subunit was cloned from the B library while ATF3 was cloned from the RP cDNA library and both were significantly recognized by BC sera. The ogenesis . The invogenesis ,73. ATF3human BC . JUND ishuman BC , suggesthuman BC ,73.Almost a century ago Otto Warburg observed that cancer cells, unlike many normal cells, rely on glycolysis rather than on mitochondrial respiration, even in the presence of oxygen. This phenomenon, confirmed by many investigators ever since, became known as the Warburg effect . WarburgGAPDH expression is associated with BC cell proliferation and with the aggressiveness of tumors . Guo et In aggregate, these reports support the view that autoimmunity plays a role in deregulating GAPDH  that migThere is evidence that PKM2, a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP, is a BC autoantigen . PKM indPKM2 is upregulated in BC tissue and high PKM2 levels were reported to be associated with poor prognosis of BC patients .Equilibrium of the PKM2 dimers and tetramers is critical for tumorigenesis and is controlled by multiple factors. As discussed above, autoantibodies in BC sera also target GAPDH, a key enzyme of glycolysis. It has been suggested that PKM2 functions independently of its pyruvate kinase activity, which is crucial for cancer cell proliferation . Hsu et In summary, the finding that this multifunctional protein and other important synergistic proteins involved in the metabolism of carbohydrates and in BC progression and metastasis such as GAPDH are targeted by autoantibodies in BC  supportsThree clones of glutathione S-transferase Pi 1 identified in the RP cDNA library  were sigGSTP1 gene seems associated with elevated BC risk in a race-specific manner [Glutathione S-transferase Pi is a susceptibility factor in esophageal and prostate cancers. GSTP1 gene polymorphism increases age-related susceptibility to hepatocellular carcinoma . Polymorc manner . c-Jun Nc manner . JNK is c manner . In suppc manner , JNK actc manner . In addic manner ,97. The c manner . The resIn summary, in addition to the recognized role of GSTP1 in detoxification and cancer susceptibility, autoantibodies to GSTP1 and to members of the AP-1 complex highly associated with the diagnosis of BC suggest that the immune system detects the participation of these signal transduction molecules in breast carcinogenesis.PINK1) gene encodes a serine/threonine protein kinase that localizes mainly to the mitochondrial inner membrane and is thought to protect cells from stress-induced mitochondrial dysfunction via the clearance of damaged mitochondria through selective mitophagy by mediating activation and translocation of PARKIN [PINK1 gene is mutated in the germ line of some patients with hereditary early-onset Parkinson’s disease (PD) [PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function [in vivo human data demonstrating that a non-coding RNA appears to ‘stabilize’ a sense coding RNA. Given the close genomic association between DDOST and PINK1 in the human genome, they speculated that these two disease related genes may be uniquely co-regulated in humans. The NAT found in BC sera  , and itsase (PD) –103. Schfunction . Althougfunction , few arefunction . Scheele BC sera  is compaBerthier et al. reported that PINK1 protein is highly expressed in epithelial tissues such as in breast carcinomas and in the central nervous system. They concluded that PINK1 displays tissue-specific subcellular location and regulates apoptosis and cell growth in BC cells, indicating that the physiologic functions of PINK1 extends beyond its regulatory role of mitochondria-mediated cell survival in neurons .PINK1 interacts with PI3-kinase/Akt signaling, mitochondrial homeostasis, and the potential context dependent pro- and anti-tumorigenic functions of PINK1 [PINK1 as a target in cancer therapeutics. Previous work by Fernández Madrid et al. [mTOR signaling, as a BC autoantigen [Paris polyphylla rhizomes. They demonstrated that polyphyllin I induces mitophagy through DRP1-mediated mitochondrial fission and apoptosis. Notably, polyphyllin I treatment stabilizes PINK1 with its accumulation at the mitochondrial surface, which recruited PARK2 and led to mitophagy. PINK1 knockdown combined with polyphyllin I treatment reduced mitophagy and enhanced DRP1-dependent fission, suggesting that PINK1 depletion leads to excessive fission and mitochondrial fragmentation [O’Flanagan and O’Neill reviewed the function of PINK1 in cancer cell biology, with an emphasis on the mechanisms by which of PINK1 . Mechaniof PINK1 . The cytd et al.  identifioantigen . The stuentation .DDOST in breast carcinogenesis. DDOST, dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit, is located in chromosome 1p36.12, a neighbor of PINK1; it encodes a component of the oligosaccharyl transferase complex that catalyzes the transfer of high-mannose oligosaccharides to asparagine residues on nascent polypeptides in the lumen of the rough ER. Enzymatic activity of this gene is involved in the processing of advanced glycation products, which develop from reactions between sugars and proteins or lipids and are associated with hyperglycemic phenotype and aging. Kim and Mak explored the hypothesis that cancer and PD may both result from PTEN dysregulation [On the other hand, little is known on the participation of gulation  and alsogulation  but the gulation , and thegulation ,113, thegulation ). Thus, PINK1 plays an important regulatory role of mitochondria-mediated cell survival in neurons in association with PD and similarly it has a stabilizing and protective effect on mitochondria outside of the nervous system. It is likely that autoantibodies targeting PINK 1 and the rearrangement involving PINK1 and its neighbor gene DDOST may be valuable biomarkers of BC risk as well as of PD.In summary, SPATA5 is a member of the AAA protein family defined by a highly conserved ATPase domain recognized by BC sera as an autoantigen  gene encodes a protein that functions in mitochondrial and peroxisomal fission and recruits fission mediator dynamin-1-like protein (DNM1L) to the mitochondrial surface [JNK-MFF signaling pathway in the carcinogenesis process [JNK-MFF signaling pathway. Inhibition of the JNK pathway and/or knockdown of MFF abolished the pro-apoptotic effect of LATS2 on A549 cells [The mitochondrial fission factor ( surface . Mitocho surface . Among t surface . Several surface . Tang et process . LATS2, 49 cells . The stu49 cells . The datThe autoreactivity of MFF  and thatA group of phages cloned from BC cDNA expression libraries exhibited inserts identical to mitochondrial proteins encoded by nDNA, one of them being MNRR1. The evidence reported by Aras et al. suggested that MNRR1 regulates multiple genes that function in promoting the cell migration required for cancer metastasis and is upregulated in cell lines derived from tumors having enhanced metastatic potential. Since MNRR1 was identified as an autoantigen in breast carcinogenesis, and other mitochondrial autoantigens are potentially downstream of MNRR1, the data presented by Aras et al. support the role of MNRR1 and mitochondrial autoimmunity in breast carcinogenesis.There is considerable evidence that the widespread autoreactivity of mitochondrial proteins found in BC sera may be the reflection of autoimmunity triggered by mitochondrial TAAs. Immune-screening of the RP cDNA library constructed with polyA RNA extracted from established BC cell lines and one pre-malignant cell, i.e., without the influence of immune cells or the society of cells found in the tumor microenvironment, led to the identification of a panel of nDNA-encoded mitochondrial proteins known to be involved in multiple tumorigenic pathways. The finding that these expression sequence tags identified in the RP cDNA library were significantly recognized by sera from patients with invasive BC indicates that the process that makes these proteins immunogenic takes place within the cancer cells themselves and constitutes direct evidence of the autoimmune nature of the reactivity.Whether the function of enzymes targeted by autoantibodies is abnormal has been debated for decades ,125. TheThe studies reviewed here demonstrate that the concept that MNRR1 directly participates in BC progression and metastasis can be extended to other autoreactive nDNA-encoded mitochondrial proteins that may contribute to mitochondrial dysfunction in BC, even if mitochondrial respiration is found to be within normal limits. Although a defect in oxidative phosphorylation may not be the cause of cancer as proposed by Warburg , the extThe data suggesting that DDX21 orchestrates a complex signaling network, with participation of JUND and ATF3 driving chronic inflammation and breast tumorigenesis, illustrate that functionally the division of mitochondrial proteins into mtDNA- and nDNA-encoded is somewhat arbitrary in this case, since DDX21 is a nucleolar protein that can translocate to the nuclei as well as to the mitochondria, whereas although JUND and ATF3 are strongly expressed in the mitochondria they are predominantly located in the nuclei.That key mitochondrial enzymes involved in energy metabolism such as GADPH and PKM2 are targeted by autoantibodies in BC makes the important point that, even if other parameters fail to show mitochondrial dysfunction, these two mitochondrial proteins are clearly involved in breast carcinogenesis. In addition, besides MNRR1, a number of other nDNA-encoded mitochondrial proteins such as GSTP1, PINK1, SPATA5 and MFF have been implicated in BC progression or BC risk, suggesting that mitochondrial autoimmunity prominently participates in breast carcinogenesis.The evidence of participation of mitochondrial autoimmunity in BC inception and progression reviewed here may challenge the prevailing thinking in oncogenesis and suggests that mitochondrial autoimmunity is a major contributor to breast carcinogenesis and probably to the inception and progression of other solid tumors.The body of work reviewed here also questions the criteria used to classify a disease as autoimmune as applied to cancer ,131. Aut