As aerobic organisms, we are continuously and throughout our lifetime subjected to an oxidizing atmosphere and, most often, to environmental threats. The lung is the internal organ most highly exposed to this milieu. Therefore, it has evolved to confront both oxidative stress induced by reactive oxygen species (ROS) and a variety of pollutants, pathogens, and allergens that promote inflammation and can harm the airways to different degrees. Indeed, an excess of ROS, generated intrinsically or from external sources, can imprint direct damage to key structural cell components  and indirectly perturb ROS-mediated signaling in lung epithelia, impairing its homeostasis. These early events complemented with efficient recognition of pathogen- or damage-associated recognition patterns by the airway resident cells alert the immune system, which mounts an inflammatory response to remove the hazards, including collateral dead cells and cellular debris, in an attempt to return to homeostatic conditions. Thus, any major or chronic dysregulation of the redox balance, the air–liquid interface, or defects in epithelial proteins impairing mucociliary clearance or other defense systems may lead to airway damage. Here, we review our understanding of the key role of oxidative stress and inflammation in respiratory pathology, and extensively report current and future trends in antioxidant and anti-inflammatory treatments focusing on the following major acute and chronic lung diseases: acute lung injury/respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis. Due to its continuously exposed surface to the external environment, the lung exhibits a formidable defense system constituted by a high number of interacting mechanisms ,2. FirstNevertheless, when one or several components of this intricate network of pulmonary defense mechanisms fail, homeostasis is disrupted, and respiratory pathology ensues causing oxidative stress and inflammation. This review briefly underscores the molecular mechanisms behind environmental stress  causing pulmonary immune-inflammatory responses and focuses on current pharmacological options, emerging, and promising therapeutic approaches and new investigational treatments targeting the most common acute and chronic respiratory illnesses.3) and nitrogen dioxide (NO2) react with several molecules at the respiratory surface and generate secondary reactive oxygen species (ROS) such as superoxide radicals (O2·−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·) [2 or ONOO−) [Environmental pollutants such as ozone (Ols (OH·) ,25. Addils (OH·) ,27,28. Fls (OH·) , or by cls (OH·) . Nitric r ONOO−) . ROS arer ONOO−) ,33. NADPr ONOO−) . Indeed,r ONOO−) ,36,37.2O2 which is detoxified by other enzymes. Catalase, decomposes H2O2 into H2O and O2, predominantly within alveolar macrophages and type II epithelial cells [2O2 or other peroxides to glutathione disulfide (GSSG) and H2O, of which GPX1 is thought to be responsible for 95% of overall lung tissue GPX activity [2O2 and protect against oxidative stress [The air–liquid interface covering the developed airways is an environment subjected to continuous oxidative stress. Accordingly, the respiratory epithelium is exposed to endogenous and also to environmental ROS. Therefore, it expresses a variety of antioxidant enzymes. Superoxide dismutases such as SOD3  ,39,40, hal cells ,43. Glutactivity . Peroxiractivity , particue stress ,47,48. Te stress ,50, catae stress ,52. Finae stress ,54.3 and NO2 [The following small non-enzymatic low-molecular-weight antioxidant molecules are highly relevant: ascorbic acid (vitamin C) , uric ac and NO2 ,61,62,63 and NO2 . Further and NO2 ,66,67,68A variety of immune and non-immune cells are activated during an inflammatory process. Each cell type releases cytokines and mediators that modify the activities of other cells, inducing an inflammatory network that progresses and resolves towards healthy homeostatic, or pathological outcomes. The lung is a vital organ for gas exchange and is constantly exposed to harmful airborne pathogens. Therefore, an immediate and intense protective/defensive inflammatory action is required to eliminate the invaders as early as possible. Nevertheless, excessive inflammation can be life threatening . ConsequThus, airborne toxicants stimulate local ROS production inducing protein oxidation, lipoxidation, glycation end products, and DNA damage, and leading to mitochondrial dysfunction, cell death, the recruitment of inflammatory cells (mainly macrophages and neutrophils), profibrotic changes or mucus hypersecretion. These oxidative stress-mediated cellular processes drive the development of key environmental respiratory diseases such as acute lung injury/respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis, and affect the progression of the most common hereditary disease affecting the lung, i.e., cystic fibrosis.Acute respiratory distress syndrome (ARDS) and its milder form acute lung injury (ALI) are critical pulmonary dysfunctions caused by heterogeneous pathologic factors  involvinARDS treatment has not experienced significant advances in the last 50 years. Early detection is the best approach to attenuate the development of ALI/ARDS. Therefore, several imaging techniques  are being assessed for the diagnosis of capillary permeability edema in early phase ARDS, indicative of damage to the alveolar-capillary membrane, given the poor sensitivity of chest X-rays ,84,85,86Asthma is a chronic inflammatory disorder of the airways in which gene–environment interactions involving a variety of cell types play an important role ,90, alth2O2 [2O2 activates mitogen-activated kinases (MAPKs) in tracheal myocytes [Oxidative stress is important in asthma ,98. Ster2O2 , oxidati2O2  and NO l2O2 . An incr2O2 . Therefo2O2 , in alve2O2 , associa2O2 ,106. Num2O2 . ROS dec2O2  and, con2O2 . ROS als2O2 ,111 and 2O2 , promote2O2 . Further2O2 ,115, dec2O2 , and sti2O2 . In addimyocytes  and stimmyocytes ,120,121.Regarding antioxidant defenses, despite increased levels of GSH in the airways, the ratio of oxidized to reduced GSH also increases . Other aConsidering treatments, among the traditional treatments are inhaled corticosteroids (ICS) ,148. TheNevertheless, even in patients with well controlled asthma, exacerbations occur. Inhaled or nebulized short-acting β2-agonists (SABAs), such as albuterol or levalbuterol, provide rapid relief of symptomatic asthma . Adding COPD is mainly initiated by inhalation of cigarette smoke, which induces oxidative damage to lung epithelial cells, leading to the development of a chronic persistent inflammatory response from the peripheral airways to the lung parenchyma . Cigaret2−) levels [ROS directly damage biological molecules and lung extracellular matrix, leading to cell dysfunction or death, and activate NF-κB, which enhances the expression of inflammatory genes such as IL-8 and TNF-α, important in COPD, contributing to reversible airway narrowing . Neutrop) levels , and lip) levels . Convers) levels . Oxidati) levels ,181. As ) levels . In cont) levels . Genetic) levels ,185,186.) levels . Increas) levels ,188, alt) levels ,142. Lik) levels ,189.Historical treatments for COPD have primarily focused on long-term oxygen therapy and restoring pulmonary function by tackling the underlying inflammation and bronchoconstriction causing air flow obstruction  . GuideliPulmonary fibrosis is the outcome of a diverse group of lung insults including toxins, fibers/particles, autoimmune reactions, drugs, and radiation. Nevertheless its primary etiology is unknown and in most cases is referred to as idiopathic pulmonary fibrosis (IPF) . AberranTilting the balance between oxidant production and antioxidant protection towards the former leads to ROS accumulation, which is promoted by fibrotic stimuli of unidentified origin ,214. In There has been no evidence for an effect of corticosteroid treatment in patients with IPF . In addiCFTR) gene. Despite the occurrence of multiorgan complications in CF, respiratory failure secondary to chronic lung infection caused by recurrent bacteria and the subsequent inflammation is the most serious outcome of this condition [−) channel at the airway apical surface cause the failure of Cl− secretion and, consequently, sodium (Na+) hyperabsorption in epithelial cells, dehydrating the airway surface fluid layer, and impairing mucociliary clearance [Cystic fibrosis (CF), the most frequent recessive hereditary autosomal disease in the Caucasian population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator  through two isoforms of NOX expressed in their apical membrane (DUOX 1 and DUOX 2) thought to be related to the CFTR channel [2O2 [2O2 and LPO either against bacteria [2O2 levels, such as TRX1, GSTP1, PRDX6 [The DUOX/LPO system is engaged by epithelial cells to produce significant quantities of ROS  provides high-level protection against the development of ALI . An alteOver the last few years, multiple biologics  have been developed targeting various participants in allergies and asthma, but mainly directed toward the complex type 2 endotype ,328. In Among anti-inflammatory treatments, antioxidant treatments stand out ,366. Vit2S) donor) decreased pro-contractile 8 isoprostane expression and modulated arginase 2 and iNOS pathways, contributing to reduced NF-κB expression in distal lung tissue. These inhibitors also mitigated eosinophil infiltration and increased tissue resistance and elastance [Thiol-based antioxidants are popular supplements to trigger GSH conversion. N-acetyl cysteine is the most commonly used thiol precursor. Thus, N-acetyl cysteine supplementation remarkably decreased inflammatory cytokines , neutrophil, and eosinophil numbers in the bronchoalveolar lavage fluid (BALF) from a mouse model , which hlastance ,389. HYDlastance . An angilastance . Diallyllastance . Mice trlastance . Pituitalastance .Furthermore, a multiple targeting approach against oxidizing molecules, PRRs and transcription factor modulators could improve outcomes in asthma. Flavonoids have stronger radical scavenging activity as compared with many other natural antioxidants because of multiple hydroxyl groups present in its chemical structure ,398,399.Thus far, no anti-IL-5 therapies have been approved for use in COPD. However, two Phase 3 studies using mepolizumab showed improvements in exacerbation frequency from subjects who had an eosinophilic phenotype and a history of COPD exacerbations, despite triple therapy . NeverthThe treatment of respiratory symptoms is the focus of most therapies for COPD, since therapies for comorbidities are ineffective or inexistent. Thus, there is a need for the identification of the mechanisms which relate COPD to its comorbidities, and preclinical models have been developed to solve this . PharmacAfter several disappointing years of promising therapies that moved into clinical trials but failed to demonstrate efficacy in IPF , the antAdministration of the senolytic drug quercetin attenuates the proinflammatory phenotype of bleomycin-induced senescence in fibroblasts . Moreove− transport to CF airway epithelia expressing CFTR mutations such as the most prevalent F508del, improving hydration and restoring mucociliary clearance [To address the most prevalent causal defects in the CFTR Cl– channel leading to CF, two biomolecular modulators are needed, i.e., CFTR correctors, to increase the amount of properly folded mutant CFTR protein at the plasma membrane, and CFTR potentiators, to allow effective gating (channel opening and closing) of the abnormal CFTR ,473,474.learance ,477. Foulearance ,479, Tezlearance , Symdekolearance  and othelearance ,490,491,Despite a thorough knowledge of the undergoing inflammatory process in CF, there are relatively few anti-inflammatory drugs in clinical use . CorticoAnti-proteases have been under investigation in CF since 1990. For example, the already described α1-antitrypsin suppressed inflammatory markers including free neutrophil elastase, proinflammatory cytokines, and neutrophils ,511. Oth2O2 and increasing GSH [Antioxidant therapies have not been yet settled in clinical practice . In factsing GSH . Of signsing GSH ,542,543,sing GSH  and ascosing GSH ,546. Onesing GSH . Alternasing GSH ,549,550,sing GSH  have yiesing GSH . AccordiAnti-inflammatory cytokines and antibodies to proinflammatory cytokines may show efficacy in CF. IL-10 possesses anti-inflammatory properties. Interferon-γ1b, another cytokine with immunomodulatory activities, did not improve pulmonary function nor alter bacterial burden or inflammatory markers in the sputum in a multicenter clinical trial, despite its ability to restore macrophage activation and antimicrobial, antiproliferative, and anti-fibrotic functions in CF cell models . To inhiP. aeruginosa [Among intracellular signaling modulators, ibuprofen (previously described), and IL-10 inhibit NF-κB activation. NF-κB activity inhibition also occurs through upregulation of peroxisome proliferator activating receptor (PPAR-γ) using PPAR-γ agonists such as thiazolidinediones (glitazones) ,557,558.ruginosa . Statinsruginosa ,561,562.Agents that augment endogenous NO production, exhibiting anti-inflammatory activity, have also been examined. In a rodent model of chronic airway infection, L-arginine, which increases NO production, was associated with reduced tissue damage, decreased neutrophil recruitment, and reduced IL-1β . SynthetIn addition, consumption of long chain fatty acids (omega-3 supplements) seems not to have a clear anti-inflammatory effect and, consequently, a positive benefit on CF disease severity .® is a complex of lactoferrin and the anion hypothiocyanite (OSCN−), with a proposed in vivo antimicrobial activity [2O2 levels in the airway surface liquid remains to be investigated. In addition, preclinical studies [−) in lung infection, which decreases BALF chemokine keratinocyte chemoattractant (KC) , IL-1β, TNF-α, and airway neutrophil infiltrate concurrent with the infectious stimulus, and enhances bacterial clearance in both wild-type and transgenic mice overexpressing the β subunit of the epithelial sodium channel Scnn1 (βENaC). Therapeutic potential of selenium-derived compounds may derive from their significant activity as inducers of phase II enzymes such as quinone reductase and GSH-S-transferase [Among emerging antioxidant and anti-inflammatory approaches for CF, targeting NF-κB with natural compounds such as resveratrol and plant extracts is highlighted. Resveratrol  is one of the most investigated natural antioxidants with a purported activity as NF-κB inhibitor. The antioxidant activity of resveratrol has been reported in several studies and also occurs in lung tissues, suggesting that resveratrol has potential as a therapeutic agent in respiratory diseases ,565,566.activity  to be ev studies  have shonsferase , increasnsferase .Because inhaled oxygen along with environmental pollutants, pathogens, and allergens, which have intrinsic oxidative potential, dissolve in the respiratory epithelial lining fluid, oxidative stress and inflammation take center stage in the wide spectrum of respiratory pathologies, a major source of disability and death following cardiovascular diseases. Thus, a timely diagnosis, treatment, as well as the management of pulmonary diseases is important. In that sense, preventive measures such as enhancement of antioxidant defenses or corrective actions to mitigate abnormal immune-inflammatory responses, whether efficacious, could provide more options for patients and clinicians. Nevertheless, the last two decades have witnessed no major breakthroughs regarding medications for most of the respiratory diseases . On the


Background: Sex-determining Region Y (SRY)-box transcription factor-2 (Sox2) belongs to the “Yamanaka's factors,” necessary and sufficient to convert somatic cells into pluripotent stem cells. In breast cancers, Sox2 expression has been associated with poor prognosis, and resistance to therapy. The aims of this study were to determine the frequency of Sox2 positivity in feline invasive mammary carcinomas (FMCs), its relationships with other clinical-pathologic variables, and with patient outcomes.Materials and Methods: This study relies on a previously described retrospective cohort of 180 FMCs, diagnosed in female cats treated by mastectomy alone, with 2-year follow-up. Sox2 (clone SP76), Estrogen Receptor alpha (ER), Progesterone Receptor (PR), Ki-67, Human Epidermal growth factor Receptor 2 (HER2), Androgen Receptor (AR), Bcl-2, Forkhead box protein A1 (FOXA1), basal markers and FoxP3-positive regulatory T cells (Tregs) were detected by automated immunohistochemistry. Sox2 expression was quantitated as an index . The FMCs were considered Sox2-positive at threshold >42%.Results: Sox2 was not expressed in the normal mammary gland or in mammary hyperplasia without atypia, but was occasionally detected in atypical hyperplasia. In FMCs, the mean Sox2 index was 38 ± 30%, and 79/180 FMCs (44%) were Sox2-positive. Sox2 expression was associated with older age at diagnosis, lymphovascular invasion, high Ki-67 proliferation indexes, low PR and FOXA1 expression, and increased numbers of tumor-associated Tregs, but was not significantly associated with the clinical stage, histological types, and histological grade. By multivariate survival analysis, Sox2 was associated with poor cancer-specific survival , independently of the pathologic tumor size, pathologic nodal stage, distant metastasis, and AR expression. A rare subgroup of FMCs characterized by an AR+Sox2–phenotype  was associated with very favorable outcomes.Conclusion: Sox2 expression was associated with poor cancer-specific survival of female cats with invasive mammary carcinomas, as previously reported in human breast cancer, but was more commonly expressed in cats than reported in breast cancers. Sox2 showed complementarity with AR in FMC prognostication. The transcription factor Sex-determining Region Y (SRY)-box transcription factor-2 (Sox2), first sequenced in humans in 1994  and humaCCND1, the cyclin D1-encoding gene, thus facilitating the G1/S transition of the cell cycle , reproductive history , medical history , the clinical tumor size and clinical stage of the mammary carcinoma at diagnosis, according to Morris , a four-The minimal length of follow-up was 2 years. Veterinary practitioners and owners of the cats of the present study were asked to provide the dates of occurrence of any local recurrences, regional spread , and distant metastases , as well as the date and cause of death. For locoregional recurrence risk analyses, censoring was “1” in case of true recurrence (at the same site as the mammary carcinoma included), new primary mammary tumor, or nodal metastasis, and “0” otherwise. The distant metastasis-free interval (DMFI) corresponded to the time period between diagnosis and distant metastasis confirmation. The disease-free interval (DFI), which is indicative of cancer progression, was censored “1” in case of locoregional recurrence and/or distant metastasis, and “0” in the absence of such events. Overall survival was censored “0” for patients still alive at the end of the follow-up period, and “1” for dead patients, whatever the cause of death. The censure applied for cancer-specific survival analyses was “1” if the animal had died from cancer, and “0” if the cat was still alive at the end of follow-up, or died from unknown cause, or died from a cause unrelated to the mammary carcinoma.Although the cases originated from two different laboratories of veterinary diagnostic pathology, they were all centrally collected at our institution . The paraffin blocks were used to generate new Hematoxylin-Eosin-Saffron (HES)-stained slides, as well as the slides for immunohistochemical analyses. Three specialists in veterinary pathology  and a MD specialist in breast cancer pathology analyzed the slides blinded to patient outcomes and reached consensus in each case.The recorded histopathological parameters were: unifocality, multifocality (multiple carcinomas within the same mammary gland) or multicentricity (multiple carcinomas in different mammary glands), the pathologic tumor size , the pathologic nodal stage , the histological type , the histological grade according to Elston and Ellis , 25, theIn this cohort, the immunohistochemical expression of ER, PR, HER2, Ki-67, EGFR, basal cytokeratins 5/6, basal cytokeratin 14, EGFR, AR, Bcl-2, FOXA1, and FoxP3 used as a regulatory T-cell marker has been previously described –21. For Statistical analyses were conducted using the MedCalc® statistical software . The threshold for Sox2 positivity (>42%) was calculated according to receiver-operating characteristic (ROC) curves that best discriminated between cats that died due to their mammary carcinoma and cats that did not die from cancer within 2 years post-diagnosis.Comparisons between Sox2-positive and Sox2-negative FMCs were done using Fisher's exact tests for categorical variables with two categories, chi-squared tests for categorical variables with more than two categories, or one-way analyses of variance for continuous variables.p-value of each covariate. A p-value <0.05 was considered significant.Univariate survival analyses relied on the Kaplan-Meier method and log-rank tests, while Cox proportional-hazards models were used for multivariate survival analyses. The results are reported using the Hazard Ratio (HR), its 95% confidence interval (95% CI), and the The immunohistochemical protocol for Sox2 detection gave a faint non-specific background staining in the cytoplasm of mast cells and in the cerebellar molecular layer. Otherwise, Sox2-specific signals were strictly nuclear. Sox2 expression was not detected at all in the normal mammary gland and hyperplasic mammary gland without atypia. Surrounding FMCs, occasional Sox2 expression was observed in mammary lobular or ductal atypical hyperplasia. A short description of Sox2 expression in other non-neoplastic feline tissues is presented in The main patient and tumor characteristics are presented in In FMCs, Sox2 expression was restricted to neoplastic cells, and not found in cancer-associated fibroblasts, tumor-infiltrating lymphocytes, endothelial cells, or any other stromal cell. The mean Sox2 index in FMCs was 38 ± 30% . Twenty-six  FMCs 14, while eCompared to Sox2-negative FMCs, Sox2-positive FMCs were diagnosed at a later age, were more likely to be associated with lymphovascular invasion and dermal infiltration, were more proliferative, and had a lower PR and FOXA1 expression . HoweverExpressed as a continuous variable (index), Sox2 expression showed positive associations with lymphovascular invasion, dermal infiltration, the Ki-67 proliferation index, and peritumoral regulatory T cells, while there were negative associations between Sox2 and PR and FOXA1 .In the 57 luminal FMCs, Sox2 was associated with a larger tumor size, more advanced clinical stage, higher histological grades, squamous differentiation, lymphovascular invasion, dermal infiltration, moderate to severe tumor-associated inflammation, higher Ki-67 indexes, and increased numbers of intratumoral Tregs, but lower PR and FOXA1 expression . There wp = 0.051). There were no significant associations between Sox2 and basal markers .In the 123 triple-negative FMCs, Sox2 expression showed a positive association with the clinical tumor size, dermal infiltration, the Ki-67 proliferation index, AR expression, and intratumoral Treg numbers . There wIn the FMCs analyzed, positivity to Sox2 was associated with shorter DMFI, DFI, and cancer-specific survival, however there were no significant associations between Sox2 expression and the locoregional recurrence risk, or overall survival. An interesting finding was that Sox2 was complementary to AR in FMC prognostication.p = 0.0398, p = 0.0288), independently of tumor size and AR expression , with tumor size, ER and AR as the covariates , independent of cancer stage at diagnosis, and independent of AR, whose prognostic value was favorable  prognostic effects in the FMCs of the present cohort, four FMC subgroups were analyzed according to AR and Sox2 expression: AR+Sox2+ , AR+Sox2– , AR–Sox2+ , and AR–Sox2– . Cox proportional-hazard models indicated however that the 3 subgroups AR+Sox2+, AR–Sox2+ and AR–Sox2– were not significantly associated with different patient outcomes (data not shown), while the AR+Sox2– phenotype was associated with excellent outcomes. This phenotype was found in 19/180 FMCs (11%), 9/57 luminal FMCs (16%), and 10/123 triple-negative FMCs (8%).Association analyses revealed that AR+Sox2– FMCs were more likely to be node-negative, diagnosed at clinical stages I–II, and free of lymphovascular invasion compared to other FMCs . AR+Sox2N = 180), the AR + Sox2– phenotype was associated with decreased locoregional recurrence risk , improved DMFI  gene, as demonstrated in the luminal MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines  is a direct ER target gene  in breast cancers, although the correlation is not significant in triple-negative breast cancers in particular   , 43. TheSOX2 expression by mammary cancer cells in a paracrine manner, while Sox2-expressing tumor cells recruit Tregs through NF- κB/CCL1 [Nuclear Factor kappa B/Chemokine (C-C motif) ligand 1] signaling . AR and Sox2 were positively associated in triple-negative FMCs only. These results suggest that luminal and triple-negative FMCs, defined in this study at ≥10% cutoff for ER and PR, are biologically distinct entities. Interestingly in human breast cancers, with cutoffs of ≥1% or ≥10% generally used for ER and PR , Sox2 asFinally, the prognostic value of Sox2 expression was investigated in FMCs, and revealed that Sox2 positivity was associated with shorter distant metastasis-free interval, disease-free survival, and cancer-specific survival. In breast cancers, Sox2 is a weak prognostic factor, but has been associated with poor disease-free survival by univariate analyses , 14, 33 ESR1 (the ER-encoding gene) overexpression has been associated with worse relapse-free survival in ER-positive breast cancer patients  in feline patients of the present study was independently modulated by Sox2, tumor size, ER and AR, with larger tumor size associated with poor prognosis, as frequently reported in FMCs , 49–55, patients , in linepatients . Indeed,patients  despite In this cohort, Sox2 and AR had independent and opposite effects on DMFI, DFI and cancer-specific survival, which triggered the analysis of FMC subgroups according to Sox2 and AR expression. The AR+Sox2– phenotype was associated with low probabilities of cancer-related death independently of tumor size, nodal and distant metastasis. Unfortunately for female cats with mammary carcinomas, this phenotype seems rare, encountered in only 11% of the cats of the present study. This rare AR+Sox2– phenotype was associated with favorable features, such as a negative nodal stage, an early clinical stage, and absence of lymphovascular invasion, low Ki-67 proliferation indexes, but high PR, FOXA1, and Bcl-2 expression. However, the AR+Sox2– phenotype was not specific of a given FMC immunophenotype, as it was found in some Luminal-A, Luminal-B, and triple-negative basal-like cases. To the authors' knowledge, AR and Sox2 have not been associated in breast cancer prognostic studies, though it could be interesting.SOX2 gene amplifications, however such amplifications are mainly reported in human serous ovarian cancers, lung squamous cell carcinomas, and glioblastomas, not in breast cancers (SOX2 is up-regulated by the transcription factor FOXO1 (Forkhead box protein O1)  , the lontein O1) , and thetein O1) .Unfortunately, a better understanding of Sox2 biology in mammary carcinomas is unlikely to have therapeutic implications in the near future. Sox2 is a poorly druggable transcription factor, but targeting signaling molecules upstream or downstream of Sox2  is under investigation in some human cancers .In conclusion, we report here that the stem cell pluripotency factor Sox2 is commonly expressed in feline mammary carcinomas, is associated with higher proliferation, decreased PR and FOXA1, but higher AR expression, as well as poor outcomes. The unfavorable prognostic effect of Sox2 was complementary to the favorable effects of AR, and independent from cancer stage at diagnosis. Understanding the mechanisms underlying Sox2 overexpression would be a prerequisite before setting eventual Sox2-targeted clinical trials in feline mammary carcinoma patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by CERVO, Comité d'Ethique en Recherche clinique et épidémiologique Vétérinaire d'Oniris, Oniris site Chantrerie, CS40706, 44307 Nantes cedex 3, France. Written informed consent was obtained from the owners for the participation of their animals in this study.FN: conceptualization, project administration, supervision, writing, and original draft preparation. YT, ED, JA, and FN: format analysis, investigation, methodology, writing, review and editing. 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.


Epigenetic modifications including DNA methylation, histone modifications, and chromatin remodeling are crucial regulators of chromatin architecture and gene expression in plants. Their dynamics are significantly influenced by oxidants, such as reactive oxygen species (ROS) and nitric oxide (NO), and antioxidants, like pyridine nucleotides and glutathione in plants. These redox intermediates regulate the activities and expression of many enzymes involved in DNA methylation, histone methylation and acetylation, and chromatin remodeling, consequently controlling plant growth and development, and responses to diverse environmental stresses. In recent years, much progress has been made in understanding the functional mechanisms of epigenetic modifications and the roles of redox mediators in controlling gene expression in plants. However, the integrated view of the mechanisms for redox regulation of the epigenetic marks is limited. In this review, we summarize recent advances on the roles and mechanisms of redox components in regulating multiple epigenetic modifications, with a focus of the functions of ROS, NO, and multiple antioxidants in plants. Epigenetic modifications refer to the mitotically- or meiotically-inheritable changes in gene expression that are not affected by the DNA sequence itself, mainly including DNA methylation, histone modifications, chromatin remodeling, and histone variants in plants and other organisms ,2. They 2O2), superoxide radical (O2•−), singlet oxygen (1O2), and hydroxyl radicals, and reactive nitrogen species, including nitric oxide (NO), peroxynitrite, nitrogen dioxide radicals. [Redox components consist of numerous oxidants and antioxidants. In plants, the primary oxidants are reactive oxygen species (ROS), for example, hydrogen peroxide . In plants, methylation occurs in the C base of “CG”, “CHG”, and “CHH”  contexts [DNA methylation typically means the specific post-replication modification over DNA molecules, in which some cytosine bases are methylated at 5′ position to become 5-methyl-cytosine  pathway, which involves 21-, 22-, and 24- nt small interfering RNAs (siRNAs) production [A. thaliana: repressor of silencing 1 (ROS1), demeter (DME), and Demeter-like 2 (DML2) and DML3 [In oduction ,26,27. Doduction ,23. Methoduction . There eand DML3 .Chromatin is the organized nucleoprotein structure in nuclei where nucleosomes are arranged. Each nucleosome is comprised of two copies of H2A, H2B, H3, and H4 histone molecules, and is wrapped by 145~147 bp double-stranded DNA . The N-tA. thaliana [Histone methylation is confined to lysine and arginine residues located at different positions of histone molecules . The transfer of methyls to histone is catalyzed by histone methyltransferase (HMT) families, which include histone lysine methyltransferases (HKMTs) and protein arginine methyltransferases (PRMTs) ,33. The thaliana ,35. Meththaliana .+ as cofactor, and other HDACs use Zn or Fe ion as cofactors. There exist 18 HDAC members in A. thaliana [Histone acetylation is the covalent modification in which acetyl groups are transferred from acetyl CoA to the epsilon-amino group of the lysine residue in histone molecules. Such modification causes the neutralization of the positive charge of the lysine, weakens the interaction between the modified histone and DNA, thus, chromatin becomes relaxed ,37. Acetthaliana ,42.The regulated change of chromatin structure is termed as chromatin remodeling. It can be changed not only by covalent modifications of histones and DNA, but also by ATP-dependent chromatin remodelers and other chromatin-associated factors. ATP-dependent chromatin remodelers are able to cause the alteration of nucleosome position, destabilization of nucleosomes or displacement of canonical histones by histone variants . EukaryoROS are byproducts of the aerobic metabolism. They act as crucial signaling molecules to mediate and integrate various growth and environmental signals to control plant development, stomatal movement, and acclimation to diverse biotic and abiotic stresses ,18,45,462O2 can easily enter the nucleus through nuclear pores, and react with nuclear proteins, including histones and transcription factors in plants [Similar to that of ROS, production of NO is an inevitable process in plant metabolism. NO is synthesized in different compartments of cells including cytosol, chloroplasts, mitochondria, and peroxisomes ,55. NO rn plants ,57,58.2•− into O2 and H2O2. CAT, APX, and GPX convert H2O2 into O2 and H2O, whereas GR is responsible for the conversion of the oxidized glutathione to the reduced one [+/NAD(P)H, GSSG/GSH, and Asc/DAsc). Their capacity to gain or lose electrons also makes them versatile carriers to alter the activities of many enzymes implicated in numerous important metabolic pathways and cellular events. NAD(P)H is vital for the transmission of redox signals. By supplying reducing equivalents to GSH and ASC through the Asada–Foyer–Halliwell cycle [2O2, and catalyze the conversion of H2O2 to H2O and O2. Under oxidative conditions, GSH-GSSG equilibrium shifts towards oxidized glutathione, leading to S-glutathionylation of proteins, an important redox-modification in plants [Cellular redox status constantly undergoes fluctuations that are balanced by different oxidant and antioxidant systems during plant development and in response to stress cues. Of the enzymatic antioxidants, SOD catalyzes the dismutation of Ouced one ,60. Of tll cycle , NAD(P)Hn plants ,63.2•− inducer paraquat increases the oxidative stress of cells. The expression of Glycerophosphodiesterase-Like (NtGPDL) is upregulated, and CG sites in the coding regions of NtGPDL are selectively demethylated [Pisumsativum suspension culture clearly decreases the global DNA methylation levels [Accumulating evidence indicates that redox intermediates govern DNA methylation levels and gene transcription in plants. In general, increases in ROS accumulation cause DNA hypomethylation. In tobacco, addition of Othylated . Similarthylated . Addition levels .Lablab purpureus plants causes the reduction in the levels of O2•− and H2O2 and the alteration of DNA demethylation and methylation levels [P. sativum [Similar to ROS, NO negatively modulates DNA methylation in plants. For instance, treatment of rice seedlings with high concentrations of sodium nitroprusside  leads to DNA hypomethylation predominantly at the CHG sites, and growth inhibition. In addition, the NO-evoked alterations in DNA methylation can be inherited to the next generation . In anotn levels . Additio sativum .A. thaliana. Activity assay showed that SAMS1 is reversibly inhibited by GSNO [In plants, three mechanisms for redox regulation of DNA methylation may exist. One mechanism is redox components modulate the synthesis of methyl donor SAM. In plants, SAM synthesis is catalyzed by S-adenosylhomocysteine hydrolase (SAHH)/homologous gene silencing 1 (HOG1), methionine synthase (MS) and S-adenosyl methionine synthase (SAMS)/methionine adenosyltransferases (MAT)  Figure . SAHH, M by GSNO . Also, p by GSNO ,72,73; aA. thaliana, two MSR genes (MSRB3 and MSRB8) are activated under high levels of NAD(H) and NADP(H), and accompanied with an increase in Met content [In the SAM cycle, the precursor of SAM is methionine (Met), which is particularly susceptible to oxidation to methionine sulfoxide (MetSo) under stress conditions . Methion content . GRXs/TR content . These iA. thaliana, impairment of folate production by sulfamethazine treatment has shown to reduce DNA methylation levels [2-THF can be interconverted by the enzyme serine hydroxymethyl transferase (SHMT). 5,10-CH2-THF can also be converted into DHF by TS. Methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase1 (MTHFD1) is also a bifunctional enzyme, and can convert 5,10-CH2-THF into 5,10-CH=THF, leading to NADPH formation. Mutation in MTHFD1 has been demonstrated to disturb folate metabolism and cellular redox state, and lead to loss of DNA methylation in A. thaliana [Arabidopsis genome, three DHFR-TS genes exist. DHFR-TS3 inhibits DHFR-TS1 and DHFR-TS2. Overexpression of DHFR-TS3 leads to decreases of DHFR and MTHFD activities, which in turn cause a drop of NADPH/NADP+ ratio [Additionally, accumulation of Met is dependent on the metabolism of folate, which provides 5-methyl-tetrahydrofolate (5-CH3-THF) for Met synthesis. In n levels . Folate n levels ,79. As sP+ ratio , and likA. thaliana, ROS mediate the irradiation-triggered DNA demethylation of bystander aerial plants. Irradiation of the roots markedly decreases the expression of DRM2, and enhances the transcriptional abundances of MET1 and DML3 in bystander aerial plants [OsCMT2 and OsCMT3 and upregulation of the DNA demethylase gene OsDME [The second mechanism for redox regulation of DNA methylation is that ROS and NO affect the expression and activities of DNA methyltransferases (DNMTs) and DNA demethylases. In l plants . Similarne OsDME . Yet, itDNA glycosylases ROS1 and DME have DNA demethylase activity. They catalyze the excision of entire methylated cytosine instead of the methyl group through the base excision repair pathway. ROS1 and DME possess Fe-S cluster assembled structure as their cofactor, and the Fe–S binding motif is essential for their enzymatic activity . Fe-S clA. thaliana, the products of DCL2, DCL3, and DCL4 are 22-, 24- and 21-nt siRNAs, respectively [A. thaliana, the activity of DCL4 is suppressed by sulfur deficiency. The DCL4 activity can be recovered by supplementation with GSH and TRXs. Moreover, immunopurified DCL4 can be activated by recombinant thioredoxin-h1 with dithiothreitol in vitro, suggesting that DCL4 is under redox regulation. Activation of DCL4 can promote 21-nt siRNA production, and may further promote DNA methylation [The third mechanism for redox modulation of DNA methylation level is that redox mediators modify the activities of dicer-like4 (DCL4) and RNAse III-like 1 (RTL1), thus affecting the production of siRNAs likely required for DNA methylation through RdDM pathway in plants ,84. siRNectively ,85. Aparectively . The rolhylation   complex, which uses NADactivity . Increasormation . An in vy in pea . Yet, wh2•− and induces PCD, followed by histone hyperacetylation due to the elevated expression of genes HAT-B and General Control Nondepressible 5 (GCN5) in maize seedlings [A. thaliana, and found that many nuclear genes are not expressed in the redox compromised state transition 7 (stn7) mutants but expressed in WT. Among these, several are epigenetically regulated. Further studies revealed that the redox signal from chloroplasts of WT rather than stn7 activates the nuclear HAT and HDAC, which promote histone acetylation and deacetylation, respectively [Arabidopsis mutants accumulated high levels of H2O2 (cat2) and were defective in GSSG to GSH conversion (gr1), showing the differential expression of GCN5-related acetyl transferase gene [Changes in redox reagents also modulate the activities of HATs and HADCs in plants . It has eedlings . Dietzeleedlings  detectedectively . Similarase gene .A. thaliana, the expression of many pathogensis-related (PR) genes is suppressed by HDA19, which deacetylates the histones on PR protein promoters under nonpathogenic condition. Pathogen attack abolishes HDA19 activity, further resulting in the acetylation of PR protein promoters and increased expression of PR-related genes [A. thaliana. The oxidation of the HDACs reduces their activity and further increases the histone acetylation of stress-responsive genes.In ed genes . Pathogeed genes . Thus, ted genes  found th+ level and NAD+/NADH ratio [+, and may further imprint on HDAC activity. In rice, NAD+-dependent sirtuin OsSRT1 has been reported to play critical roles in suppressing glycolysis by deacetylating histones and glyceraldehyde-3-phosphatedehydrogenase [+ level.In plants, the activities of sirtuin HDACs are dependent on the NADDH ratio . Thus, orogenase . Redox mA. thaliana. S-nitrosylation has stronger effects than S-glutathionylation on the HDAC activity. In addition, SA has been shown to induce endogenous NO generation, which represses HDAC activity and stimulates histone acetylation [DlHD2 and two ethylene-responsive factor-like genes DlERF1 and DlERF2 enhances during longan fruit senescence. Treatment with NO delays the fruit senescence, elevates the transcription of DlHD2, but diminishes the expression of DlERF1 and DlERF2. These data imply that NO modulates fruit senescence possibly through affecting the expression of HD gene in longan [A. thaliana, the HD2 type member HDT2 (histone deacetylase 2) and HDT3 have been identified to be S-nitrosylated [Mengel et al.  found thtylation . Moreoven longan . Additioosylated .A. thaliana [OsDDM1a and OsDDM1b, as well as DNA hypomethylation [A. thaliana [A. thaliana, topoisomerase VI (Topo VI) A subunit (AtTOP6A), a chromatin-associated factor, has been demonstrated to mediate singlet oxygen signals from the plastid to the nucleus. Under 1O2 accumulation condition, AtTOP6A binds to the promoters of 1O2-responsive AAA-ATPase gene and a set of other 1O2-responsive genes, and directly activates the expression of these genes. Topo VI also regulates the transcription of H2O2-responsive genes under high light stress. However, changes in the expression of 1O2- and H2O2-responsive genes modulated by AtTOP6A are different, suggesting that Topo VI is capable of integrating multiple signals produced by ROS in plants under stress [DNA methylation 1 (DDM1) is an important SWI/SNF2 chromatin remodeler, and can shift nucleosome composition and mediate DNA methylation by allowing MET1, CMT2, and CMT3 to access DNA, especially in heterochromatin regions in plants . Mutatiothaliana . In ricehylation , indicatthaliana . It is ithaliana , suggestr stress .Redox mediators, particularly ROS and NO have been emerging key regulators of chromatin remodeling in plants. They greatly influence not only the transcription and activities of multiple enzymes, catalyzing the addition or removal of methyl and acetyl groups in DNA and histones, but also the biosynthesis and supply of methyl and acetyl donors to DNA and histones. Redox-regulated changes in the epigenetic marks shape chromatin organization, further controlling the expression of many genes and other molecular processes, thereby profoundly affecting plant growth and stress responses. In recent years, much progress has been made on the roles of redox mediators in regulating DNA methylation, and histone modifications in plants. However, many reported actions of redox components on epigenetic marks are indirect effects, and the precise molecular mechanisms underlying the processes are largely unknown. Whether epigenetic modification changes are caused by one oxidant without triggering other antioxidants is also poorly described. Moreover, the research works on redox regulation of other epigenetic marks like phosphorylation, ubiquitinylation, glycosylation, ADP-ribosylation, and sumoylation of histone, chromatin remodeling, and siRNA are quite limited to date.It has been documented that pathogen attack and diverse abiotic stresses significantly modify epigenetic marks ,8,9. ROS


Hydrangea luteovenosa is a member of the family Hydrangeaceae. The complete chloroplast genome sequence of H. luteovenosa was characterized from MiSeq (Illumina Co.) pair-end sequencing data. The chloroplast genome of H. luteovenosa was 157,494 bp in length with a pair of inverted repeats (IRs)  which were separated by a large single-copy (LSC)  and a small single-copy regions (SSC) . It contained 129 genes including 85 protein-coding genes, 36 tRNA genes, and 8 ribosomal RNA genes. The maximum-likelihood phylogenetic analysis with the previously reported chloroplast genomes showed that H. luteovenosa is most closely related to the tribe Hydrangeeae. Hydrangea (Hydrangeaceae) consists of 23 species that are mainly distributed in eastern Asia and northern America .The study for http://www.macrogen.com/kor/), we assembled the chloroplast genome using Geneious R10.0.4 . A map of the chloroplast genome was illustrated using OGDRAW  was 157,494 bp in length and contains a pair of inverted repeat (IR) regions of 25,126 bp, large single copy (LSC) region and small single copy (SSC) region with the lengths of 86,596 bp and 18,646 bp, respectively. It contained 129 functional genes including 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. There were 16 protein-coding genes, 12 tRNA, and 8 rRNA genes, which were duplicated in the IR regions. The LSC region contained 61 protein-coding and 21 tRNA genes, whereas 11 protein-coding and 1 tRNA genes were included in the SSC region. Fourteen genes contained one or two introns including the protein-coding genes, atpF, clpP, ndhA, ndhB, petB, petD, rpl2, rpoC1, rps12, rps16 and ycf3. The base composition is asymmetric  with an overall GC of 36%. Phylogenetic analysis was performed using chloroplast coding gene sequences of H. luteovenosa and those of 14 related species of Hydrangeaceae family including Loasa tricolor . Total genomic DNA was extracted from ∼100 mg of fresh leaves using a DNeasy Plant Mini Kit . After generating the pair-ended reads using the MiSeq platform (Illumina Co.) at Macrogen (tipetala .


Malignant pleural mesothelioma (MPM) is a rare and aggressive malignancy that most commonly affects the pleural lining of the lungs. It has a strong association with exposure to biopersistent fibers, mainly asbestos (80% of cases) and—in specific geographic regions—erionite, zeolites, ophiolites, and fluoro-edenite. Individuals with a chronic exposure to asbestos generally have a long latency with no or few symptoms. Then, when patients do become symptomatic, they present with advanced disease and a worse overall survival (about 13/15 months). The fibers from industrial production not only pose a substantial risk to workers, but also to their relatives and to the surrounding community. Modern targeted therapies that have shown benefit in other human tumors have thus far failed in MPM. Overall, MPM has been listed as orphan disease by the European Union. However, molecular high-throughput profiling is currently unveiling novel biomarkers and actionable targets. We here discuss the natural evolution, mainly focusing on the novel concept of molecular epidemiology. The application of innovative endpoints, quantification of genetic damages, and definition of genetic susceptibility are reviewed, with the ultimate goal to point out new tools for screening of exposed subject and for designing more efficient diagnostic and therapeutic strategies. Simian Virus 40 (SV40) infection was previously explored as aetiologic agent, but its role was not definitively proven -Gene-NCBI: Pubs; 2016. http://www.ncbi.nlm.nih.gov/pubmed/ (accessed on 1 December 2020)). It encodes for a 90 kDA deubiquitinating enzyme (DUB), which regulates key cellular pathways, including cell cycle, cellular differentiation, transcription, and DNA damage response [BAP1 germline mutations has been described in two families with high MPM incidence [BAP1 allele, whereas inactivation of the second allele occurs later in life. In animal models, BAP1+/− mice exhibited significantly increased incidence and rate of progression of asbestos-induced MPM than wild type models [BAP1+/− mice carried bi-allelic inactivation of the BAP1 gene coherently to its predisposing role as a cancer susceptibility candidate gene. From comprehensive profiling of MPM high-risk families, BAP1 germline mutations emerge as generally associated with less aggressive disease and a 3.5/7-fold improved survival, while in other cancer types, they are associated with poor prognosis. Moreover BAP1-mutated MPM most frequently feature epitheliod differentiation and predominant peritoneal involvement, and arise in female patients; the age of disease onset is overall lower than that in wild type patients and similar to that registered in other hereditary cancer syndromes [BAP1 who harbor more than one type of primary cancer, and this observation strongly suggests that multiple tissue lineages are affected by BAP1 deficiency [BAP1 loss has been consistently reported in approximately 50% of all sporadic mesotheliomas. In vivo studies have demonstrated that the occurrence of germline BAP1 mutations in combination with asbestos exposure determine a significant increased incidence of MPM and more aggressive tumors, pointing out a synergy between genetic and environmental factors. It has been suggested that individuals carrying mutations in BAP1 should avoid asbestos exposure, cigarette smoking, as well as excessive sun exposure. The identification of BAP1 as a marker of familial cancer syndromes allowed for the design of BAP1-pharmacologic blockade strategies. Due to their role in homologous recombination repair [BAP1-null tumor cells with contrasting results. In a first report, no differential sensitivity was observed between BAP1-WT and BAP1-mutant MPM cells treated with the MK4827 (Merck) inhibitor [BAP1-null cells as compared to wild-type and heterozygous BAP1-null cells [BAP1, which leads to the loss of 12 amino acids within the catalytic and BARD1 binding domains [BAP1 status [BAP1 can transcriptionally promote the expression of HDAC2. The BAP1 knockdown led to a decrease in HDAC2 but an increase in HDAC1 expression. This imbalance seems to play a role in promoting MPM cell sensitivity to the histone deacetylase vorinostat [BAP1 loss and EZH2 upregulation. Specific EZH2 inhibitors  decreased cell proliferation and reduced invasion and clonogenic capacity in mesothelioma cell lines and in in vivo experiments [For any given level of exposure to a carcinogen, not all the exposed subjects develop the disease . In thischanisms . The BAPresponse . The genncidence . Germlinncidence . Similare models . Indeed,yndromes . This obyndromes ,44. Inteficiency ,46,47. Bn repair , poly, which is a cyclin-dependent kinase inhibitor, and p14(ARF), which binds the p53-stabilizing protein MDM2 (www.omim.org/entry/600160 (accessed on 1 December 2020)). Somatic CDNK2A mutations frequently occur in sporadic MPMs. However, in a report by Betti and colleagues, in two out of six families affected by both mesothelioma and melanoma, the authors showed BAP1 germline nonsense mutation or a recurrent pathogenic germline mutation  in the CDKN2A gene [CDNK2A mutations were categorized as “low asbestos exposure”, without occupational exposure. This observation allowed the authors to hypothesize that CDKN2A germline mutations could lead to increased risk for developing MPM. Germline mutations in the TP53 gene have also been reported. It is well known that germline TP53 mutations are causatively associated with Li–Fraumeni syndrome [TP53 mutations associated with peritoneal malignant mesothelioma in women patients [TP53 germline missense change (pArg213Gln) was found in a large family characterized by the occurrence of different cancer types, among which MPM in a subject who did mention exposure to asbestos was found [NF2 gene have been reported. It encodes for merlin, a tumor suppressor protein frequently inactivated in borderline tumors such as schwannoma and meningioma [BAP1 signaling pathway. For example, analysis of 407 pleural mesothelioma cases and 389 controls with a comprehensive history of asbestos exposure revealed an increased risk of abnormalities in chromosomal region 7p22.2, which includes the gene encoding the Forkhead box protein K1 (FOXK1) that interacts with BAP1 [BAP1 that confer differential susceptibility to mesothelioma development and treatment response. Recent studies have identified novel susceptibility candidates. In Western Australia, a large study (428 cases and 1269 controls) reported variants located in the CRTAM, SDK1, and RASGRF2 genes that were significantly associated with MPM risk [Although more rarely described, deleterious missense mutations in other genes have been found in familial clusters of mesotheliomas. The N2A gene ,44. Patisyndrome . Massivepatients ,56. Furtas found . Finallyningioma . The seqningioma . Howeverith BAP1 . HoweverMPM risk . In thosMPM risk .p21), 9 (9p21), and 22 (22q12); number losses ; gains (5); or a combination  [BAP1 gene located at 3p21.3 site have been reported in 20–25% of sporadic MPM cases [BAP1 expression were common in sporadic MPMs, in the absence of germline changes. This evidence confirms that BAP1 behaves as a tumor suppressor. Interestingly, the occurrence of BAP1 mutations/deletions seems to be an early event in MPM onset as demonstrated by extensive whole-exome sequencing analysis of in situ MPMS [CDKN2A and NF2. Exon sequencing analysis on platinum/pemetrexed-treated MPM revealed somatic changes in 23 genes including BAP1, TP53, NRAS, and EGFR, as well as increased copy number of the CDKN2A and CDKN2B genes [CUL1), phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta gene (PIK3C2B), TAO kinase 1 gene (TAOK1), and radixin gene (RDX) [RAPGEF6 and ACTG1 genes are mainly reported in non-epithelioid subtypes [Several acquired chromosomal alterations have been identified in MPM, including deletions in chromosome 3 (3 and 22) . SomaticPM cases ,40,48, aPM cases ,63,64. Titu MPMS ; they aritu MPMS . Massive2B genes . Somaticne (RDX) ; changessubtypes . Overallsubtypes . Indeed subtypes  and autosubtypes . The prosubtypes . Upon assubtypes . Within subtypes .NF-2) gene. The latter acts as tumor suppressor that is part of the NF-2/Merlin complex that makes up the NF2/Hippo pathway. Overall, patients with NF-2 mutation had a worse prognosis [NF-2 and BAP1 to ferroptosis, a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides [BAP1 inhibits the expression of the scavenger SLC7A11 [BAP1 single nucleotide variants and more frequent loss of chromosome 22 q. The non-epitheliod subtypes carry more frequent CDKN2A deletions in men. Overall, in the analyzed cohort, the loss of CDKN2A is associated with shorter survival and in male patients. Otherwise, TP53 mutations have been detected more frequently in females [SETD2, TP53, NF2, and ULK2) as the most significantly mutated genes featuring significant differences among cluster 1 (sarcomatoid MPM) and cluster 2–4 , thus suggesting that the differences observed in overall survival in cluster 1 might be related to the differential combination of gene alterations. Busacca et al. demonstrated on tissue samples differential miRNA signatures between biphasic-sarcomatoid and epithelioid-sarcomatoid tumors, whereas no differences in terms of histotypes were found with regards to the expression of hsa-miR-31, hsa-miR-221, or hsa-miR-222 [The occurrence of somatic changes in MPM genome that, as showed above, are caused by multiple mutational processes, generates characteristic mutational signatures . Among trognosis . Recent eroxides ,79. In f SLC7A11 . Thus, a SLC7A11 . Further females . Interes females  allowing-miR-222 . Among t-miR-222 ,84; no iInformation on gene expression and modification are gaining increasing relevance in molecular epidemiology studies since it reflects both genetic and environmental influences . EpigeneTMEM30B, KAZALD1, and MAPK13 methylated genes) and has been suggested to be predictively associated to MPM development [SFRP2 and SFRP5 tumor suppressor genes, which encode secreted frizzled-related proteins, has been observed in MPM samples. Promoter methylation profile of these genes could potentially serve as a plasma-based epigenetic biomarker [BAP1 in mice resulted in increased H3K27me3 and elevated expression of EZH2. Furthermore, pharmacological inhibition of EZH2 in BAP1-mutant MPM cell lines blocked their growth, and findings in preclinical models have suggested a relationship between BAP1 and activation of EZH2 [Epigenetics refers to the heritable changes beyond the DNA sequence, which are reversible and include different processes such as DNA methylation, chromatin modifications, nucleosome positioning, and alterations in non-coding RNA profiles. Epigenetic mechanisms modulate gene expression and thus influence disease onset and progression. For this reason, it results in a focus of molecular epidemiologic investigation . To thiselopment . The epiiomarker . Moreoveiomarker . Moreoveiomarker  and autoiomarker . HGBM1 aiomarker . Subsequiomarker . Notablyiomarker . Other diomarker , which iiomarker . In addi of EZH2 . In stro of EZH2 . More im of EZH2 ,105.miR 17-92 cluster and its paralogs, namely, miR 17-5p, 18a, 19b, 20a, 20b, 25, 92, 106a, and 106b to be markedly upregulated in MPM if compared to normal mesothelial cell lines [let-7e, miR-7-1, miR-9, miR-34a, miR-144, miR-203, miR-340, miR-423, and miR-582, and a higher expression of let-7b, miR-30b, miR-32, miR-195, miR-345, miR-483-3p, miR-584, miR-595, miR-615-3p, and miR-1228 have been detected in MPM tissue samples compared with normal cells [miR-101-3p and miR-494 have been found to be downregulated in chronic pleuritis and pleural hyperplasia, whereas the same study reported miR-181a-5p, miR-101-3p, miR-145-5p, and miR-212-3p expression to be reduced in transformed tissues [miR-103 have been reported to be significantly decreased in MPM patients vs. health subjects [miR-30b, miR-32, miR-483-3p, miR-584, and miR-885-3p target tumor-suppressor genes such as CDKN2A, RB1, and NF2. The overexpression of miR-29c has been reported to behave as a tumor suppressor and mediates epigenetic effects, leading to downregulation of DNMT1 and DNMT3A genes [miR-31 expression is related to the deletion of the miR-31 gene located in chromosome 9p21.3, which is commonly altered in aggressive MPMs. Indeed miR-31 loss is associated the development of both chemo- and radioresistance in MPM cell lines [miR-9, miR-7-1, and miR-203 target EGF, HGF, JUN, and PDGFA oncogenes. Interestingly, miRNa expression has been tested in pleural effusion as well. This approach is of particular interest for MPM with potential prognostic and therapeutic implications. In 2019, Birnie and colleagues investigated the microRNA profile of neoplastic pleural effusion compared with inflammatory one and found that the combination of miR-143, miR-210, and miR-200c could significantly differentiate MPM [miR-548a-3p and miR-20a were potential diagnostic markers for MPM [miR-16-5p, miR-103, miR-98, miR-148b, miR-744, and miR-30e-3p, and these signatures present diagnostic accuracy and can be also exploited for therapeutic purposes [miR-31 expression in MPM and reactive mesothelial proliferations has demonstrated that it is significantly upregulated in transformed mesothelioma, specifically in sarcomatoid subtypes [miR101, miR25, miR26b, miR335 and miR433; two miRNAs were found to be downregulated , and two miRNAs were expressed exclusively in patients (miR29a and miR516) [miRN-146A rs2910164 polymorphism has been significantly associated with risk of MPM onset since carriers of two polymorphic alleles features lower risk of disease development [MSLN) gene could be a putative miRSNP and that MSLN is a candidate target of miR-611 and, to a lesser extent, of miR-887 [From the different cell types, miRNAs can be secreted into the extracellular space and then transported to the circulating blood flow and in body fluids as pleural effusions . The vasll lines . Moreoveal cells . miR-101 tissues . The levsubjects . The maj3A genes . For insll lines . On the iate MPM . Predict for MPM . The aut for MPM . Malignapurposes ,122. Comsubtypes . Differe miR516) . Polymor miR516) . Several miR516) . Differeelopment . Within  miR-887 .http://www.rarecarenet.eu/ (accessed on 1 December 2020)). That is why in MPM there is a strong rationale to exploit molecular epidemiology in discovering and validating potentially actionable markers. Novel therapeutic approaches are summarized in The recent improvement in the knowledge of molecular mechanisms leading to pleural malignant transformation has allowed for the start of novel and more promising tailored therapeutic approaches . MPM is p14ARF and the p16INK4A genes. The resulting genetic defects are defined by inactivation of the p53-mediated pathways and progression of cell cycle. Reintroduction of defective gene should be performed through an adenoviral vector bearing the p14 or the p16 gene , suppresses the cell proliferation through pRb dephosphorylation, and reactivates the p53-mediated checkpoint mechanisms. (ii) Gene therapy targeting signal transduction. A main interesting example is related to the observation that MPM is characterized by overexpression of VEGF and VEGFR1 and two pathways, as well as the HGF–MET pair [NK4 gene [interferon-β gene can activate CD 8 T cells against MPM when delivered locally into the pleural space. Another study showed that MV is an appropriate vector for immunotherapy when used in combination with anti-PD-L1 or anti-CTLA-4 antibodies, therefore having potential application in MPM therapy [To date, the management of MPM is still complex, and patient survival remains poor. However, most promising therapeutic programs are emerging on the basis of the more recent advances in molecular characterization and evolution of the disease. Although a detailed description of all the novel therapeutic approaches goes beyond the scope of this paper MET pair . The NK4MET pair . NotablyMET pair . Thus, iNK4 gene . (iii) ONK4 gene . OverallNK4 gene . VirotheNK4 gene . It has  therapy .BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). It has been demonstrated that BAP1 loss is associated with increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets [BAP1-inactivationevaluated in an open-label, phase 2 trial. Overall TAZ showed antitumor activity in BAP-1-eficient MPM patients and was generally well tolerated [BAP1 stabilizes BRCA-1 and promotes poly-(ADP-ribose)-dependent recruitment of polycomb deubiquitylase complex PR–DUB to DNA damage sites [BAP1 isoform lacking part of the catalytic domain sensitized MPM cells to the PARP1 inhibitor, olaparid, and this sensitivity could be augmented by concomitant treatment with apitolisib (GDC-0980), a dual inhibitor of class 1 PI3K, and mTOR kinase, which downregulates BRCA-1.The lack of efficacy of DNA hypomethylating agents in solid tumors has been related to their high systemic toxicity, mainly myelosuppression. Clinical efficacy of vorinostat, a histone deacetylase inhibitor, has been investigated in a phase 3 trial, as a second-line or third-line therapy improved patients’ outcome. Quite surprisingly, vorinostat did not improve OS  and could not be recommended as a therapy for patients with advanced cases . Very in targets . Recent olerated . Moreovege sites . BAP1 ismiRNA pharmacogenomics can be defined as the study of miRNAs and polymorphisms affecting miRNA function in order to predict drug behavior and to improve drug efficacy. A growing body of literature is emerging related to the potential application of microRNA-based therapy. Preclinical studies have pointed out that re-expression of miR-145 , miR-126omics domains, shifting the clinical paradigm towards real personalized and preventive healthcare. Therefore, besides the traditional clinical variables  stage), new -omics entities, such as radiomic or radiogenomic signatures, will probably be introduced in clinical practice in the near future, allowing for the establishment of revolutionary treatments that pave the way towards a better care in mesothelioma patients. However, technologic improvements have brought about the biological and clinical complexity of this disease when facing the overwhelming number of variables to be consider in prevention, diagnosis, and treatment decisional processes. This traditional approach  is quickly reaching its limits and also becoming economically unsustainable.As well what has been discussed above, MPM also represents an enduring challenge for physicians. Both the diagnostic process and the therapeutic protocols have reached high levels of complexity by integrating innovative variables from complex -This ongoing revolution has also precipitated concerted efforts by many investigators to define molecular subgroups of mesothelioma, characterize the genomic landscape of different subtypes, identify novel therapeutic targets, and define mechanisms of sensitivity and resistance to targeted therapies. Therefore, besides histological, clinical, and demographic information, a wide range of data obtained from genomics, proteomics, immunohistochemistry, and imaging should be integrated by physicians to improve patient survival on the basis of more effective stratification tools, namely, a precision medicine approach.Such a multiomic approach will provide physicians with an enormous amount of data (“big data”). Apart from analyzing and interpreting them individually, the task to integrate these rapidly expanding non-structured data will soon become too complicated for one medical doctor.In this framework, with the advent of artificial intelligence (AI), nowadays physicians potentially have the adequate technology to deal with precision medicine approaches. Indeed, AI excels in recognizing complex patterns in medical data and provides a quantitative, rather than qualitative, assessment of clinical conditions. AI methods are precise and allow for specific quantification of features not otherwise quantifiable by human experts. The growing complexity of the human–machine and human–software interactions in conjunction with the increasing incidences of cancer have created a workforce shortage throughout the world. Furthermore, the knowledge and experience gap between more developed and under-resourced healthcare environments poses an enormous public health challenge and represents one of the great global inequities in cancer care.omics techniques to MPM is at the beginning, and limitations are related to its rarity, the nonspecific symptoms, and the long latency of arousal after asbestos exposure. Otherwise, it could represent an exciting strategy to improve the knowledge on pathogenic mechanisms on one hand, and on novel therapeutic agents and tools on the other.A multidisciplinary approach to MPM has allowed for the development of molecular and genetic epidemiology. This approach is emerging as a powerful tool for this rare and aggressive disease for diagnostic, prognostic, and therapeutic purposes. Although there is a strong interconnection among different expertise and know-how across clinics, basic science, biostatistics, and bioinformatics are required to properly collect and process samples; to analyze and validate biomarkers; as well as to design large-scale screening, monitoring, and risk studies. The application of -


The endoplasmic reticulum (ER) is an interconnected organelle that plays fundamental roles in the biosynthesis, folding, stabilization, maturation, and trafficking of secretory and transmembrane proteins. It is the largest organelle and critically modulates nearly all aspects of life. Therefore, in the endoplasmic reticulum, an enormous investment of resources, including chaperones and protein folding facilitators, is dedicated to adequate protein maturation and delivery to final destinations. Unfortunately, the folding and assembly of proteins can be quite error-prone, which leads to the generation of misfolded proteins. Notably, protein homeostasis, referred to as proteostasis, is constantly exposed to danger by flows of misfolded proteins and subsequent protein aggregates. To maintain proteostasis, the ER triages and eliminates terminally misfolded proteins by delivering substrates to the ubiquitin–proteasome system (UPS) or to the lysosome, which is termed ER-associated degradation (ERAD) or ER-phagy, respectively. ERAD not only eliminates misfolded or unassembled proteins via protein quality control but also fine-tunes correctly folded proteins via protein quantity control. Intriguingly, the diversity and distinctive nature of E3 ubiquitin ligases determine efficiency, complexity, and specificity of ubiquitination during ERAD. ER-phagy utilizes the core autophagy machinery and eliminates ERAD-resistant misfolded proteins. Here, we conceptually outline not only ubiquitination machinery but also catalytic mechanisms of E3 ubiquitin ligases. Further, we discuss the mechanistic insights into E3 ubiquitin ligases involved in the two guardian pathways in the ER, ERAD and ER-phagy. Finally, we provide the molecular mechanisms by which ERAD and ER-phagy conduct not only protein quality control but also protein quantity control to ensure proteostasis and subsequent organismal homeostasis. Post-translational modification (PTM) by ubiquitin (ubiquitination) is a very powerful and finely tuned process that is spatiotemporally involved in nearly all aspects of biological and physiological functions for the maintenance of organismal homeostasis. Comprehensive proteomics studies have identified tens of thousands of ubiquitination sites on thousands of proteins, indicating that the vast majority of cellular proteins are ubiquitinated during their lifetime. Among the enzymes catalyzing ubiquitination—including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases—E3 ubiquitin ligases govern the efficiency, complexity, and specificity of ubiquitination, since E3 ubiquitin ligases participate in the specific recognition of signals present on substrate proteins ,5,6,7. IIn the endoplasmic reticulum (ER), folding of proteins is inherently error-prone. Even with an elaborate network to ensure adequate protein folding and assembly, misfolding and subsequent aggregates of proteins can occur quite frequently. To maintain protein homeostasis, referred to as proteostasis in the ER, eukaryotes have evolved multiple quality control mechanisms involving ER-associated degradation (ERAD), ER-phagy, Golgi quality control, and plasma membrane quality control . While mIn this review, we first conceptually outline ubiquitination machinery and catalytic mechanisms of E3 ubiquitin ligases. We then highlight the mechanistic insights into the E3 ubiquitin ligases involved in the protein quality and quantity control processes at the ER. We finally discuss both protein quality control and quantity control at the ER, which are mainly mediated by ERAD and ER-phagy.UBB, UBC, RPS27, and UBA52, encode ubiquitin in mammals. Genes UBB and UBC, as polyubiquitin cassettes, encode linear fusions of three and nine ubiquitin molecules, respectively, while RPS27A and UBA52 encode ubiquitin as in-frame fusion to small and large ribosomal proteins, respectively, from which deubiquitinating enzymes (DUBs) cleave off the monomeric ubiquitin . ,22. 8,22 enzymes ,25,26,27 enzymes . Upon biter bond .The E1 ubiquitin complex transfers ubiquitin to the E2 ubiquitin-conjugating enzyme through a transthioesterification reaction . There aE3 ubiquitin ligases finally associate with both the E2 ubiquitin complex and a substrate protein to catalyze the covalent transfer of ubiquitin, thereby leading to the formation of an amide bond of ubiquitin, most commonly with a lysine residue or the N-terminus of the substrate . InteresDUBs reverse ubiquitination and switch off the ubiquitin signal, which deals with the matched complexity of ubiquitination. In humans, more than 100 DUBs have been identified. DUBs consist of six structurally distinct families of different cysteine and metalloproteases. The five families of cysteine proteases are the ubiquitin-specific proteases (USPs) with 54 members, the ubiquitin C-terminal hydrolases (UCHs) with four members, the ovarian tumor proteases (OTUs) with 16 members, the Josephin family with four members and the motif interacting with ubiquitin (MIU)-containing novel DUB family (MINDYs) with four members ,41,42,43Despite the mechanism of catalysis being conserved in all eukaryotes, the network of ubiquitin is more sophisticated in mammals due to the large number of E2 ubiquitin-conjugating and E3 ubiquitin ligase enzymes, compared to only two E1 ubiquitin-activating enzymes. There are approximately 40 E2 conjugating enzymes and 700 E3 ligases in humans that function in numerous combinations to ubiquitinate virtually every protein in the cell . E3 ubiqE3 ligases are divided into three main families on the basis of the structure of the ubiquitin-charged E2 conjugating enzyme-binding domain and the ubiquitin transfer mechanism  5,9,33],9,335,9,With approximately 30 members in the human genome, homologous to the E6-associated protein (E6AP) carboxyl terminus (HECT)-type E3 ligases contain not only a C-terminal HECT domain that forms a thioester intermediate with ubiquitin but also various N-terminal domains that govern substrate specificity and cellular localization, both of which adopt a characteristic bilobal structure with a short hinge that enables the lobes to rotate and supports ubiquitin transfer . On the PARK2 gene mutated in Parkinson’s disease), the central E3 subunit of the linear ubiquitin chain assembly complex (LUBAC), two RING fingers and DRIL 1 (TRAID1), and human homologue of Ariadne (HHARI), all of which are highly conserved from yeast to humans [RING-in-between-RINGs (IBR)-RING (RBR)-type E3 ligases, as a unique family of RING–HECT hybrid E3 ligases, are characterized by a homologous sequence, consisting of two predicted RING fingers, RING1 and RING2, and a central IBR zinc-binding domain . Additioo humans ,74,75,76o humans .In spite of a plethora of structurally unrelated proteins, their ubiquitination is highly selective. Substrate specificity and type of polyubiquitin chains are conferred by E2 conjugating enzymes in the process of RING- and RBR-type E3 ligases-mediated ubiquitination, whereas they are governed by E3 ligases in the process of HECT-type E3 ligases-mediated ubiquitination, suggesting the importance of E2 conjugating enzymes, E3 ligases, and combinations of these two enzymes ,66,78.E4 ubiquitin ligases play roles in highly processive chain elongation but not in the initial steps of ubiquitination ,81,82,83p300 and CREB-binding protein (CBP) possess E4 ligase activity in their N-terminal regions ,93,94. TThe C-terminus of the Hsc70-interacting protein (CHIP) possesses not only one U-box domain but also three tandem tetratricopeptide repeat (TPR) motifs that bind to Hsc70 and Hsc90 . SimilarYin-Yang 1 (YY1) is an E4 ligase and facilitates p53 polyubiquitination by enhancing the interaction between p53 and MDM2 . FurtherUbiquitination determines the fate of substrate proteins. Functions of ubiquitination are dictated by their distinct structural topologies that are recognized by specific ubiquitin adaptors. Monoubiquitination, modification by a single ubiquitin moiety, is the most abundant modification and controls protein recognition, complex formation, allosteric regulation, endocytosis and even proteasomal degradation ,100. FurAlthough the functional significance of K48- and K63-linked polyubiquitination is largely known, the biological significance of K6-, K11-, K27-, K29-, K33-, and M1-linked polyubiquitination is still far from being fully understood. Four ubiquitin molecules linked via K48 are enough to target substrate proteins to proteasomal degradation and K63-linked polyubiquitin chains serve as signals for modulation of protein trafficking, DNA repair and signal transduction ,110,111.Interestingly, heterotypic conjugates potentially lead to combinatorial control of signaling. K11/K48-branched chains function as proteasomal priority signals and target aggregation-prone and cytotoxic proteins to proteasomal degradation ,115. AddDespite an elaborate network within the ER for co- and post-translational folding and maturation of polypeptides, the process of protein folding and maturation is inherently error-prone and therefore misfolded proteins must be eliminated ,122,123.3Man9GlcNAc2  is first appended to consensus asparagine residues within canonical N-glycosylation sites (NxS/T), which plays a leading part in monitoring conformational maturation, directing correctly folded proteins to the ER exit and directing misfolded proteins to ERAD [1Man9GlcNAc2 generated by cleavage of three terminal glucose residues from the core glycans, thereby monitoring the folding of glycoproteins. To specifically generate a glycan signal for ERAD, terminally misfolded proteins must escape from the calnexin or calreticulin, which is conducted by mannosidases—including ER mannosidase I (ERManI) [Substrate recognition, the commitment step for ERAD, is accomplished by molecular chaperones and chaperone-like lectins . During  to ERAD . The gly(ERManI) ,137, ER (ERManI) ,140,141,(ERManI) —that pro(ERManI) ,144. The(ERManI) ,146,147.(ERManI) ,149. Add(ERManI) ,151.Energy-dependent retrotranslocation of substrates from the ER back into the cytoplasm is essential for ERAD, since the ER does not possess the components of UPS such as the E1 activating enzyme, E2 conjugating enzyme, and the proteasome . ImportaProtein quality control E3 ubiquitin ligases involved in ERAD determine the specificity, plasticity, and efficiency of ERAD and subsequently fine-tune proteostasis. As unrestrained degradation would destroy cellular homeostasis, it is crucial that E3 ligases ensure only the specificity required to eliminate misfolded proteins, while leaving their correctly folded counteracts intact. Moreover, E3 ligases usually need to target similarly misfolded and potentially cytotoxic factors, suggesting the necessity of a delicate balance between specificity and plasticity. Approximately 40 ERAD E3 ubiquitin ligases have been identified in mammals and three in yeast to date . HoweverSeveral ERAD E3 ubiquitin ligases, including glycoprotein 78 (gp78), also known as autocrine motility factor receptor (AMFR)), hydroxymethylglutaryl reductase degradation protein 1 (HRD1), membrane-associated RING C3HC4 finger 6 (MARCHF6), and RING finger protein 5 (RNF5), are transmembrane proteins ,169,170,Interestingly, for the degradation of ERAD substrates, mixed K48/K11-linked polyubiquitin chains are appended, facilitating the association with downstream components of ERAD machinery . FurtherHRD1-mediated ERAD is the most conserved and best characterized branch of mammalian ERAD and it is indispensable for versatile homeostatic processes, including energy metabolism, food intake, systemic water balance, and immune cell development ,191,192.B cell development-specific pre-B cell receptor (pre-BCR), B-lymphocyte-induced maturation protein 1 (BLIMP1), Fas, inositol-requiring enzyme 1 (IRE1), NF-E2-related factor 2 (NRF2), p53, peroxisome proliferator activated receptor γ coactivator-1 β (PGC1β), and pro-opiomelanocortin (POMC) have been identified as endogenous substrates for SEL1L/HRD1-mediated ERAD ,208,209.A variety of studies have suggested HRD1 as a critical immune regulator. HRD1-mediated ERAD eliminates major histocompatibility complex (MHC) class I heavy chains that fail to acquire their native conformation in complex with β2-microglobulin and peptide, suggesting the role of HRD1 as a positive regulator for the peptide antigen presentation by MHC I molecules ,213,214.HRD1 is involved in anti-apoptosis . HRD1-meAlthough the underlying mechanisms by which HRD1 recognizes nuclear substrates remain elusive, HRD1 may directly mediate the degradation of nuclear transcription factors, including BLIMP1, NRF2, and PGC1β ,215,219,Gp78 is another major ERAD E3 ubiquitin ligase in mammals . Five trGp78 plays an essential role in fine-tuning the levels of proteins quantitatively in response to specific signals. Sterol accumulation alters transmembrane regions of 3-hydroxy-3-methyl-glutaryl-coenzyme A (CoA) reductase (HMGCR), which is an ER membrane-anchored protein, and catalyzes the rate-limiting step of sterol biosynthesis, in which HMG-CoA is reduced to mevalonate ,226 and It has been suggested that Gp78 has neurobiological functions. Gp78 mediates ubiquitination and subsequent degradation of disease-associated aggregative proteins, such as superoxide dismutase-1 (SOD1), ataxin-3, and the mutant form of huntingtin ,229. FurInterestingly, gp78 also functions as an E4 ubiquitin ligase in a cooperative manner with other ERAD E3 ubiquitin ligases . For exaMARCHF6, akin to Doa10 in yeast, is a large multi-pass E3 ubiquitin ligase embedded in the membranes of the ER and plays a central role in sterol and lipid metabolism ,232,233.HMGCR and squalene epoxidase  are controlled by MARCHF6-mediated ERAD, although SQLE is widely considered as the canonical substrate for MARCHF6 ,241. SQLMARCHF6 itself has a short half-life due to its autoubiquitination. Interestingly, USP19, an ER-resident DUB, and cholesterol enhance the stability of MARCHF6 ,251.RNF5 is a membrane-anchored E3 ubiquitin ligase localized not only in the ER but also in mitochondrial membranes ,169,170.p97/VCP functions as a bridge to connect retrotranslocated substrates to cytoplasmic cofactors for further processing and subsequent degradation of ERAD substrates, suggesting that p97/VCP is closely associated with the proteasome-mediated degradation of substrates . CytoplaRetrotranslocated ERAD substrates should be rapidly eliminated to prevent the formation of protein aggregates in the cytoplasm. Bag6, ubiquitin-like protein 4A (Ubl4A), and the transmembrane domain recognition complex 35 (Trc35) co-chaperone small glutamine rich TPR-containing protein α (SGTA) form a chaperone complex, which prevents the formation of protein aggregates . Bag6 olWhile most of the misfolded proteins in the ER are efficiently eliminated via ERAD, the pore size of the retrotranslocon may preclude misfolded oligomeric or aggregated proteins from being exported from the ER, which leads to the development of another mechanism, selective autophagic degradation of the ER, referred to as ER-phagy, which eliminates these toxic substrates, thereby maintaining proteostasis. The term ER-phagy was first used upon the discovery of ER whorls, which occur after unfolded protein response (UPR)-associated lipid synthesis in yeast ,263. ER-The identification of versatile ER-phagy receptors responsible for substrate recognition in mammals, including family with sequence similarity 134 member B (FAM134B), reticulon domain containing family member 3 long (RTN3L), secretory 62 homologue (SEC62), cell-cycle progression gene 1 (CCPG1), atlastin 3 (ATL3), and testis-expressed protein 264 (TEX264), has advanced our understanding of ER-phagy ,270,271.Since the discovery of sequestration of the ER by autophagosomes over 50 years ago , it has Several functional characteristics of the ER-phagy receptors have been reported . The ER-FAM134B, also known as reticulophagy regulator 1 (RETREG1), is classified as an IM ER-resident protein characterized by the presence of LIR and RHD . RHD medRTN3L, a long splice isoform of RTN3, is an ER tubules resident protein. RTN3L possesses C-terminal RHD for anchorage of RTN3L to the ER from the cytosolic face of the membrane. RTN3L possesses an extended N-terminus containing six distinct LIR motifs spaced at uneven intervals and oligomerizes to engage and cluster LC3, thereby leading to the formation of autophagosomes at the ER tubules ,291. RTNCCPG1 is a type II single-pass transmembrane protein and possesses both LIR- and focal adhesion kinase family interacting protein of 200 kDa (FIP200)-interacting regions, which associate with LC3 and FIP200, respectively . In contTEX264 is a single-pass transmembrane protein and possesses a negligible N-terminal luminal region of approximately five amino acids and a C-terminal cytosolic LIR ,271. TEXIn addition to the transmembrane ER-phagy receptors, p62 could also mediate ER-phagy. In mouse liver, xenobiotics-induced excess ER is eliminated by p62-mediated autophagy . IRE1 asMicro-ER-phagy was first discovered as piecemeal micronucleophagy in yeast . AdditioIn mammals, micro-ER-phagy is involved during recovery from cyclopiazonic acid-induced ER stress, in which ER-derived vesicles are directly engulfed by endolysosomes . CompareVesicular delivery, a pathway for lysosomal degradation of ER-derived single-membrane vesicles, has been recently proposed ,281. CalIn the last decade, multiple studies have implicated the pivotal roles of the ER in the progression of neurodegenerative diseases, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Gaucher disease (GD) ,309,310.2+ homeostasis, thereby leading to the induction of ER stress. α-SYN aggregates activate sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in neurons, which results in alteration of ROS production and calcium metabolism and cell death [PD is a progressive neurodegenerative disease and is characterized by the aggregates accumulation of misfolded α-synuclein (α-SYN) fibrils into proteinaceous inclusions in Lewy bodies (LB) or Lewy neurites . Toxicitll death ,323.PARKIN, an E3 ubiquitin ligase, is involved in a large variety of cellular processes related to PD ,325. IntHRD1 attenuates 6-hydroxydopamine (6OHDA)-induced cell death in dopaminergic neuroblastoma cells .2+ release through ryanodine receptors (RyRs) and subsequent neuronal cell death [2+ signaling and autophagy defects in a GD mouse model, suggesting the relation between Ca2+ and autophagy in GD [GD is the most common lysosomal storage disorder (LSD) and is an autosomal recessive sphingolipidosis caused by mutations in a lysosomal enzyme, glucocerebrosidase (GCase), or its activator protein, saposin C, which functions in hydrolysis of glucosylceramide (GlcCer) to ceramide and glucose ,329. Thell death ,331. Intgy in GD ,333.Misfolded mutants of GCase are retained in the ER and are not efficiently transported from the ER to the lysosomes, which are subject to ERAD ,335. IntThe ER is an interconnected organelle that plays fundamental roles in the biosynthesis, folding, stabilization, maturation, and trafficking of secretory and transmembrane proteins. It is the largest organelle and critically modulates nearly all aspects of life, including food intake, water balance, growth, metabolism, and immunity. Therefore, the ER undergoes continuous reconstruction to maintain its function and integrity. Unfortunately, even with an elaborate network to ensure adequate protein folding and assembly, misfolding and subsequent formation of protein aggregates can occur quite frequently, which could be a primary pathogenic mechanism in multiple human disorders. Therefore, mammals have developed two sophisticated guardian pathways, ERAD and ER-phagy, for the maintenance of proteostasis and organismal homeostasis. However, the relative contributions of these two guardian pathways remain still elusive. Many proteins can be eliminated by both pathways, suggesting that ERAD and ER-phagy are not mutually exclusive ,246,337.The field of ubiquitin has been extensively studied in the last several decades and a large number of studies aim to improve our understanding of the complex nature of the ubiquitin network. Given that E3 ligases are dysregulated in various processes of pathogenesis, E3 ligases have emerged as promising therapeutic targets for the treatment of diseases. Compared to ERAD, the involvement of specific E3 ubiquitin ligase and ubiquitination during the process of ER-phagy remains largely unknown. p62 has been shown to engage ubiquitinated cargos with autophagosomes during ER-phagy . More reDeregulation of protein quality control as well as protein quantity control at the ER and the subsequent failure in the re-establishment of proteostasis are closely associated with various human diseases, including cancer, cardiovascular diseases, immune diseases and neurodegenerative diseases ,120. The


Scientific Reportshttps://doi.org/10.1038/s41598-020-61253-2, published online 09 March 2020Correction to: This Article contained errors, whereby two affiliations were omitted for the author András Gézsi. The correct affiliations for András Gézsi are listed below:Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1089, HungaryMTA-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Semmelweis University, Budapest, HungaryDepartment of Measurement and Information Systems, Budapest University of Technology and Economics, Budapest, HungaryIn addition, the Figure Legend for Supplementary Videos 1 and 2 were omitted from the Supplementary Information 3 file. The correct legend for these videos can be seen below:“Two video presentations of the 3D structures of the in vivo circulating NETs from unstimulated cell-free plasma shown in figure 2.”These errors have now been corrected in the HTML and PDF versions of this Article, and in the accompanying Supplemental Information 3 file.