Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are characterized by an inflammatory response, alveolar edema, and hypoxemia. ARDS occurs most often in the settings of pneumonia, sepsis, aspiration of gastric contents, or severe trauma. The prevalence of ARDS is approximately 10% in patients of intensive care. There is no effective remedy with mortality high at 30–40%. Most functional proteins are dynamic and stringently governed by ubiquitin proteasomal degradation. Protein ubiquitination is reversible, the covalently attached monoubiquitin or polyubiquitin moieties within the targeted protein can be removed by a group of enzymes called deubiquitinating enzymes (DUBs). Deubiquitination plays an important role in the pathobiology of ALI/ARDS as it regulates proteins critical in engagement of the alveolo-capillary barrier and in the inflammatory response. In this review, we provide an overview of how DUBs emerge in pathogen-induced pulmonary inflammation and related aspects in ALI/ARDS. Better understanding of deubiquitination-relatedsignaling may lead to novel therapeutic approaches by targeting specific elements of the deubiquitination pathways. For severe COVID-19 patients, ALI/ARDS represent one of the major pathological changes; phenotypes include inflammatory infiltration and inflammatory storm, alveolar epithelial–capillary damage, lung embolism and hemorrhage, hypoxia, and poor prognosis with high mortality. The pathobiology of the disease is incompletely understood [Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are a group of illnesses with features of lung inflammation, air–blood barrier disfunction, and hypoxemia. ALI/ARDS are life-threatening with a severe public health concern, approximately 200,000 people per year develop into ALI/ARDS in the United States, and the mortality rates are high at 30–40% ,2,3,4,5.derstood ,7,8. FurOverwhelmed immune responses are believed to be a major contributing factor in the pathogenesis of ALI/ARDS. In the initial pulmonary infection, invaded microbial pathogens including viruses and bacteria attract and activate residential microphages to release chemokines and cytokines, along with infiltration of leukocytes, particularly neutrophils and lymphocytes, into the alveolar sacs ,9. In idIn the meantime, the invasion of microbial pathogens causes airway epithelial and pulmonary endothelial cell death, destroys alveolar architecture, and damages the air–blood barrier ,12. ThesProteins dynamically exert their diversified functions in life processes in response to different pathophysiological settings. In concert with gene transcription, ubiquitin proteasome degradation governs the abundance and availability of the protein in the cell. Most of the proteins modified by a post translational modification called ubiquitination are deemed to be degraded ,18. UbiqSaccharomyces cerevisiae [The process of ubiquitination is reversible, a group of enzymes called deubiquitination enzymes conduct the enzymatic process  Figure . Deubiqurevisiae ,25,26. Arevisiae . Five farevisiae . The sixrevisiae . The catrevisiae . The phyrevisiae . TherefoMounting studies have focused on inflammation to dissect its underlying molecular mechanisms in the pathogenesis of ALI/ARDS. Deubiquitinating enzymes play crucial roles in modulation of inflammation by changing the protein stability of the critical molecules . SeveralAlveolar residential macrophages are central to the development of the inflammatory response by recruiting neutrophils and circulating macrophages to the site of injury, their functions are modulated by deubiquitinating enzymes ,97. ThesPseudomonas aeruginosa bacterial load, probably via interferon signaling in bronchial lung epithelial cells [The activities of deubiquitinating enzymes are involved in many aspects of the pathogenesis in ALI/ARD. Lung epithelial cell death is a hallmark in ALI/ARDS. Massive lung epithelial cell death has been reported in ARDS patients. Lung epithelial cell death is regulated by deubiquitinating enzymes. Loss of DUB CYLD can activate NF-κB to inhibit apoptosis in lung epithelial cells . In lungal cells . OTUB1 ial cells . Furtheral cells ,52. In aal cells . Finallyal cells . For exaal cells . This stUntil now, the mechanism of COVID-19 infection has not been well illustrated yet. From the biopsy or autopsy of COVID-19 patients, diffuse damage of lung parenchyma has been shown ,103. ExpStreptococcus pneumoniae infections by inhibiting plasminogen activator inhibitor-1 (PAI-1) expression [S. pneumoniae-induced nuclear factor of activated T cells (NFAT)signaling pathway by deubiquitinating TGF-β-activated kinase 1(TAK1) [Escherichia coli pneumonia with enhanced NF-κB activation [The USP subfamily contains the majority of DUBs encoded by the human genome, which are the most diversified members within the DUB family ,111,112.pression ,48. Furt 1(TAK1) . In conttivation . Perhapstivation . These dUSP7 (HAUSP)is originally identified as a viral binding protein that preferentially cleaves K11-, K63- and K48-linked ubiquitin chains ,120. USPUPS13 is also reported to regulate antiviral responses, however, its function is controversial. USP13 is considered to promote IFN signaling and play an antiviral role by stabilizing STAT1 . NeverthThe OTU family DUBs can be divided into four subfamilies, including OTULINs (OTULIN and FAM105A), OTUBs/Otubains (OTUB1 and OTUB2), OTUDs , and A20s  . The majOTUB1 and OTUB2 regulate virus-triggered IFN inflammation by deubiquitinating TRAF3 and TRAF6 . OTUB1 sOTUD1 knockout mice show more resistance to virus infection and LPS stimulation ,83. OTUDThe JAMMs are the third largest subfamily in DUBs, and it comprises 12 members: COP9 signalosome subunit (CSN)5, 26S proteasome non-ATPase regulatory subunit 14 (POH1), BRCA1/BRCA2-containing complex subunit 3 , MPN domain containing , eukaryotic translation initiation factor 3 subunit (EIF3)H, CSN6,26S proteasome non-ATPase regulatory subunit 7 (PSMD7), EIF3F, anti-Müllerian hormone (AMSH), AMSH-LP, and pre-mRNA-processing-splicing factor 8 (PRPF8) ,138,139.The enzymes of the UCH protein family includes four members, UCHL1/PGP9.5 (protein gene product 9.5), UCHL3, UCHL5/UCH37, and BRCA1 associated protein-1(BAP1), which contain a conserved catalytic UCH domain of ~230 amino acids ,143. TheThe MJD family onlycontains four members: Ataxin (ATXN)3, ATXN3L, Josephin domain containing (JOSD), and JOSD2 . StudiesThe MCPIP, also known as ZC3H12A (zinc finger CCCH-type containing 12A) family includes MCPIP1-7 members ,147,148.Like the recently identified DUBs, the MINDY family contains four members: MINDY1–4 , which aPathogen-related DUBs are promising potential targets of drug discovery for human pathogen infection and associated inflammatory disorders. Bacteria-encoded DUBs might promote bacterial pathogenicity through inhibiting the human ubiquitin–proteasome system . FurtherDuring the past decade, studies began to dissect the role of DUBs in ALI/ARDS. Increasing evidence proved that immune responses, inflammation, cell death, air–blood barrier integrity, and invasiveness of the pathogens are fine-tuned by DUBs in ALI/ARDS . Modulat


Liver diseases represent a critical health problem with 2 million deaths worldwide per year, mainly due to cirrhosis and its complications. Oxidative stress plays an important role in the development of liver diseases. In order to maintain an adequate homeostasis, there must be a balance between free radicals and antioxidant mediators. Nuclear factor erythroid 2-related factor (Nrf2) and its negative regulator Kelch-like ECH-associated protein 1 (Keap1) comprise a defense mechanism against oxidative stress damage, and growing evidence considers this signaling pathway as a key pharmacological target for the treatment of liver diseases. In this review, we provide detailed and updated evidence regarding Nrf2 and its involvement in the development of the main liver diseases such as alcoholic liver damage, viral hepatitis, steatosis, steatohepatitis, cholestatic damage, and liver cancer. The molecular and cellular mechanisms of Nrf2 cellular signaling are elaborated, along with key and relevant antioxidant drugs, and mechanisms on how Keap1/Nrf2 modulation can positively affect the therapeutic response are described. Finally, exciting recent findings about epigenetic modifications and their link with regulation of Keap1/Nrf2 signaling are outlined. Liver diseases represent a critical health problem with 2 million deaths worldwide per year, mainly due to cirrhosis and its complications as a result of damage induced by chronic and excessive alcohol consumption, obesity, hepatitis B virus (HBV), and hepatitis C virus (HCV), which represent the main etiological factors for liver damage development ,2. SeverNrf2 is a pivotal player in the preservation of cellular homeostasis. In liver, Nrf2 has an important protective role during inflammation, fibrogenesis, and carcinogenesis . In normOn the other hand, several studies have suggested that epigenetic changes, defined as heritable changes to gene expression without affecting DNA sequence, can regulate Keap1/Nrf2 activity ; DNA metThe aim of this review is to provide an up-to-date overview of the Keap1/Nrf2 signaling pathway and its involvement in the development of main liver diseases such as alcoholic liver damage, viral liver diseases, steatosis, steatohepatitis, cholestatic damage, and liver cancer. In addition, the main current pharmacological therapies and epigenetic modifications related to this transcription factor will be described. Development of liver damage is characterized by necrosis of parenchymal cells, exacerbated inflammatory response, and alterations in the composition of the extracellular matrix (ECM) . At the ROS include molecules derived from oxygen reduction, such as molecules or atoms that have unpaired electrons and are unstable and highly reactive because they can react with adjacent molecules subtracting electrons. Oxidative intermediaries can generally be classified into oxygen-centered radicals, such as superoxide radical, hydroxyl radical, and peroxyl radical, and oxygen-centered non-radicals, such as hydrogen peroxide and singlet oxygen . ProductIn the liver, mitochondria, cytochrome metabolism, microsomes, and peroxisomes are the main endogenous sources of ROS, which, in turn, can produce harmful effects on lipids, proteins, and DNA, altering their functions . MoreoveAntioxidants are agents that postpone or prevent the oxidation of oxidizable substrates and scavenge ROS. The human body has different mechanisms to counteract oxidative damage, and these defenses can be both enzymatic and non-enzymatic . EnzymatThe liver is the primary organ responsible for the metabolism of xenobiotics, and for this reason, it possesses an extensive and varied set of antioxidant defense mechanisms. The Keap1/Nrf2 pathway is the major system responsible for maintaining liver homeostasis when this organ suffers oxidative damage . Nrf2 reKeap1, the main intracellular regulator of Nrf2, is a 624-aminoacid protein that functions as an adapter for Cul3-Rbx E3 ubiquitin ligase complex, and is composed by five domains: a N-terminal domain, a broad complex, tramtrack and bric-a-brac (BTB) domain, an intervening region (IVR), a double-glycine repeats (DGR) domain, and a C-terminal domain, each with an important role in Nrf2 activity inhibition ,17. NotaIn normal conditions, Nrf2 is located in the cytoplasm bound to its inhibitor protein Keap1 and is rapidly degraded by the ubiquitin-proteasome pathway . HoweverInactivation of Nrf2 is mediated mainly by Keap1, which facilitated the poly-ubiquitination of Nrf2 by the Cullin 3-Ring box protein (Cul3-Rbx) complex, and consequently Nrf2 is degraded by the 26S proteasome . Human KAlternatively, Nrf2 activity can be controlled by a proteasomal degradation mechanism mediated by the serine/threonine protein kinase glycogen synthase kinase 3 (GSK-3) and the E3 ligase adapter β-TrCP . β-TrCP 2O2 as a product of fatty acid oxidation [2O2 decreased the expression of Peroxisome proliferator activated receptor alpha (PPARα) as well as its target genes Carnitine palmitoyltransferase 1A (CPT-1) and Acyl-CoA oxidase (ACOX) that are involved in fatty acid oxidation. Additionally, H2O2 upregulates the expression of sterol regulatory element-binding protein-1c (SREBP-1c), with a consequent increase in fatty acid synthase (FAS), thus promoting lipid accumulation [MAFLD includes NASH, fibrosis, cirrhosis, and hepatocellular carcinoma ,27. Clinxidation , and a smulation . The indmulation . Endoplamulation . ER-stremulation . In addimulation . In the mulation . Patientmulation . On the mulation . Becausemulation , it is aIn subjects with MAFLD, Nrf2 activation occurs in response to disease development, but the expression of antioxidant enzymes seems to decrease as MAFLD progresses . Nrf2-knThe p62 protein or sequestosome 1 (SQSTM1) protects against lipotoxicity through activation of the non-canonical Keap1/Nrf2 pathway in hepatocytes; in the absence of p62, cells are most susceptible to oxidative stress . AutophaNrf2 activation ameliorates methionine- and choline-deficient (MCD) diet-induced hepatic steatosis, inhibiting Cluster of differentiation 36 (CD36), Fibroblast growth factor 21 (Fgf21), and PPARα expression in the liver of mice , whereasLactobacillus mali APS1 reduced the levels of hepatic lipids by regulating the expression of sirtuin-1 (SIRT-1)/peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α)/SREBP-1, and it also increased the hepatic antioxidant activity via induction of Nrf2 and HO-1 [ob/ob mice showed a reduction in Glut4 protein levels and in Akt phosphorylation [The expression of Nrf2 was elevated in livers of mice fed an HFD for 12 weeks . In contand HO-1 . Nrf2-KOand HO-1 . Similarand HO-1 . In contand HO-1 . Nrf2 neand HO-1 ,46,53. Cand HO-1 , and CD3and HO-1 ,54. Howerylation . In contrylation . Mice wirylation . Interesrylation .To date, there is no clear explanation for the aforementioned discrepancies in the role of Nrf2 during diet-induced obesity. The observed apparent contradictions could be due to differences in experimental design, including the gender, genetic background, and age of the mice, as well as the mechanism(s) involved in Nrf2 activation in the various settings.Oxidative stress is the main component of hepatocellular injury and may worsen inflammation and fibrosis in the liver of patients with NASH . AdditioNrf2 plays a role key in NASH, and its activation has been found to protect against NASH ,9,60. Nrc-met and Keap1 (in which Nrf2 is overactivated) and fed an MCD diet for 4 weeks, displayed increased liver mass but decreased triglyceride deposition [Nrf2 activation exerts potent anti-inflammatory effects , and Nrfposition .Green tea extract and ezetimibe have been found to promote the protective effect of Nrf2 against lipid deposition and inflammation in NASH through p62-dependent activation of Nrf2 . The herLactobacillus plantarum activates the Keap1/Nrf2 pathway, and promotes the activities of SOD, CAT, HO-1, and GSTK1, which reduced oxidative stress in the liver induced by alcohol [Alcoholic liver disease includes simple fatty liver (steatosis) and more severe forms of liver damage, including alcoholic steatohepatitis, cirrhosis, and HCC . Oxidati alcohol . Finally alcohol .Studies in Nrf2-KO mice demonstrated most convincingly the protective role of Nrf2 against ethanol-induced damage. In mice treated with ethanol, loss of Nrf2 was shown to result in severe steatosis, liver inflammation, and mortality . Lastly,Hepatic fibrosis is a common reversible wound-healing response to chronic liver injury and inflammation and is mainly attributed to HSC activation that occurs after long-term liver damage by alcohol, HBV, HCV, and non-alcoholic fatty liver. When aggravated, hepatic fibrosis can progress to cirrhosis and hepatocarcinoma . 4)-induced liver inflammation and fibrosis [Overexpression of Nrf2 in hepatocytes of c-met/Keap1 KO mice, resulted in less oxidative stress, inflammatory cells, and fibrosis moderately reduced . In contfibrosis ,41. Ligufibrosis .4 treatment showed prolonged inflammatory and profibrogenic responses. In a different model, sulforaphane suppressed hepatic fibrosis induced by bile duct ligation (BDL) in mice [Increased Nrf2 expression in response to ginsenoside Rg1 and chebulic acid resulted in inhibitory effects on HSC activation and experimental hepatic fibrosis ,71. Phar in mice . In summOxidative stress, UPR, and lipid peroxidation are associated with the pathogenesis of liver cirrhosis . The mRNCholestasis is characterized by impaired hepatic bile flow, which leads to accumulation of bile acids and other chemicals in liver and blood. BDL, a model of extrahepatic obstructive cholestasis, causes induction of inflammatory liver injury and fibrosis . In addiNrf2-KO mice with BDL showed reduced GSH excretion, and higher levels of intrahepatic bile acids, Mrp3 and Mrp4, whereas bile acid synthetic enzymes CYP7a1 and CYP8b1 were decreased . In contKEAP1 were identified in multiple cohorts of HCC [NFE2L2, the gene that encodes Nrf2, are more common than in Keap1; these mutations occur as late events in HCC, as they were found in advanced stages of human liver carcinogenesis [HCC, one of the most common tumor types in the world, accounts for more than 80% of all hepatic malignancies . The incs of HCC . In HCC,ogenesis .NFE2L2 or KEAP1 activate the Keap1/Nrf2 pathway, increasing the nuclear abundance of Nrf2 and promoting the subsequent activation of its target genes, which results in tumor cell survival and promotion of tumorigenesis [KEAP1 were less sensitive than wild-type cells to short- and long-term treatment with sorafenib. KEAP1 inactivation led to sorafenib, lenvatinib, and regorafenib resistance in HCC cells through induction of Nrf2 target genes and reduction of ROS levels [Mallory–Denk bodies (MDBs) and intracellular hyaline bodies (IHBs) have been described as cytoplasmic inclusions in a subtype of HCC. MDBs are composed of the intermediate filament proteins keratin 8 (K8) and K18, as well as p62 and ubiquitin, whereas IHBs consist of p62 and/or ubiquitin. The presence of IHBs was found to be associated with significantly shorter overall survival in patients with HCC . Severaligenesis . Evidencigenesis . Nrf2 inigenesis . Nrf2 ovigenesis . Finallyigenesis . Severaligenesis . Moreoveigenesis . In addiS levels . Other cS levels . TherefoHCV may cause chronic hepatitis, steatosis, fibrosis, cirrhosis, and HCC. Several studies correlated cellular stress with HCV infection, and persistence of oxidative stress during HCV replication has been widely observed . The HCVThere are conflicting results related to interference of HCV with the Keap1/Nrf2 pathway. In HCV-infected cells, Nrf2 activation is mediated through mitogen-activated protein kinases, casein kinase 2, phosphoinositide-3 kinase, and protein kinase C, thus contributing to cell survival against HCV infection . In contChronic infection with HBV causes liver inflammation and can promote fibrosis, which can ultimately lead to cirrhosis and HCC ,89. ProtThere are different compounds with antioxidant capacities that can protect cells from intrinsic and extrinsic cellular stress and that have also shown efficacy in the treatment of liver diseases. These compounds typically act with a great affinity for ROS, donating electrons to counteract their reactivity and maintaining the redox balance . Many plThe polyphenol RSV is a phytochemical present in fresh grape skin, red wine, peanuts, and berries . DiffereCurcuma longa, which has several pharmacological properties as an antioxidant, anti-inflammatory, anti-fibrogenic, anti-microbial, and anti-carcinogenic compound [4 [Curcumin or diferuloylmethane is a natural polyphenol obtained from rhizomes of compound . As an acompound . Curcumipound [4 ,103; it pound [4 . Curcumipound [4 . Novel dpound [4 . In clinpound [4 . In a clpound [4 . Curcumipound [4 .O-methyl quercetin (3′MQ) and quercetin3-O-glucuronide (Q3GA), have shown cytoprotective effects against alcoholic liver damage [Quercetin is a flavonoid present in high concentration in apples and onions; it possesses various pharmacological properties, acting as an antioxidant, anti-inflammatory, bacteriostatic, cardioprotective, and anti-carcinogenic compound . Besidesr damage .H)-pyridone, is an antifibrotic, anti-inflammatory and antioxidant molecule used for the treatment of idiopathic pulmonary fibrosis [PFD or 5-methyl-1-phenil-2--1-2-dithiole-3-thione is a synthetic dithiolethione that activates Nrf2 and promotes the transcription of several antioxidant genes. This drug promotes GSH biosynthesis and the expression of enzymes related with phase II biotransformation such as NQO1 . In in vminophen . The bilminophen ; it can minophen ,124. Finminophen . Studiesminophen . Other aminophen .While all compounds reviewed in this section have shown efficacy and safety in experimental studies and a few of them have also been evaluated in clinical trials with good results, clinical use is often hampered by suboptimal pharmacokinetic properties, as discussed.Epigenetics emphasizes mechanisms that regulate gene expression without modifying the DNA sequence. DNA hypermethylation or hypomethylation and histone modification have important roles in gene expression and activity of Keap1/Nrf2 signaling .Nrf2 promoter inhibits mRNA expression; however, when cells are incubated with the demethylating agents 5-Aza and TSA, the activity of DNA methyltransferase 1 (DNMT1) and histone deacetylase (HDAC) is inhibited, restoring the mRNA expression levels of Nrf2. Similarly, in humans, methylation of 3 CpG sites has been reported to inhibit Nrf2 expression in prostate cells [Keap1 gene promoter causes Nrf2 overactivation and Nrf2 nuclear accumulation; such responses have been observed in breast, kidney, colon, lung, and head and neck cancer tissues [Several authors have postulated that epigenetic regulation of Nrf2 is a possible therapeutic target in the treatment of diseases related to oxidative stress or drug metabolism. For example, Yu et al.  used murte cells . The poste cells ,130,131. tissues .Keap1 promoter, which upregulates its transcription and suppresses the Nrf2 response [Histone modifications also have an impact on the Keap1/Nrf2 pathway. Deacetylation of H3 and H4, mediated by HDAC and HDAC2, decreases Nrf2 signaling and the antioxidant response and increases the sensitivity to oxidative stress . Similarresponse ,7.The aforementioned epigenetic mechanisms, as well as the antioxidant molecules involved in modulating the activity of the Keap1/Nrf2 signaling pathway, are summarized in Regardless of its etiology, the spectrum of chronic liver disease involves different damage processes including oxidative stress, which can be a trigger for inflammation and fibrosis; these can ultimately lead to more severe stages of liver damage, such as cirrhosis or cancer. In recent years, interest has arisen in studying the transcription factor Nrf2 as a potential therapeutic target for the treatment of liver diseases, including epigenetic modifications that favor the activity of this endogenous antioxidant system. At present, it is known that different antioxidant molecules such as pirfenidone, curcumin, quercetin, etc., exert their effects by modulating the Nrf2 pathway; however, few studies have elucidated in detail the molecular modifications exerted by these drugs specifically in the liver.Due to the complex pathophysiology of liver diseases, there is currently no effective treatment to counteract this damage. It is hoped that studying the antioxidant properties of various compounds can make them good candidates for the treatment of these diseases, especially by focusing beyond their roles as ROS scavenger, and specifically on their effects as modulators of Nrf2 signaling and/or as modulators of epigenetic mechanisms.


METΔex14) allows for the selection of specific kinase inhibitor treatment in patients with non-small cell lung cancer (NSCLC). Multiplex technologies are recommended in this setting. We used nCounter, a multiplexed technology based on RNA hybridization, to detect ALK, ROS1, RET, and METΔex14 in RNA purified from cytological specimens (n = 16) and biopsies (n = 132). Twelve of the 16 cytological samples (75.0%) were evaluable by nCounter compared to 120 out of 132 (90.9%) biopsies. The geometrical mean (geomean) of the housekeeping genes of the nCounter panel, but not the total amount of RNA purified, was significantly higher in biopsies vs. cytological samples. Among cytological samples, we detected ALK (n = 3), METΔex14 (n = 1) and very high MET expression (n = 1) positive cases. The patient with METΔex14 had a partial response to tepotinib, one of the patients with ALK fusions was treated with crizotinib with a complete response. Cell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using RNA-based methods such as nCounter.The detection of ALK receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping ( METΔex14 splicing variant associate with responses to EGFR and MET tyrosine kinase inhibitors, respectively (EGFR- and MET-TKIs) [Although lung cancer continues to be one of the tumors with the highest mortality rate in the world, in recent years, the identification of specific alterations in various genes has allowed the development of targeted therapies, which have improved the clinical outcome of selected groups of patients. Mutations in the EGFR gene and the ET-TKIs) ,3, whileET-TKIs) .MET alterations were not assayed [The need to test all markers associated with therapeutic options and the fact that a significant number of patients with non-small cell lung cancer (NSCLC) only have cytological samples available for molecular diagnosis makes urgent the development of multiplexed techniques that can detect clinically relevant markers with a minimum amount of sample. The most widely used multiplexed methodologies for the detection of mutations, amplifications, and fusions in formalin-fixed, paraffin embedded (FFPE) biopsies are DNA and RNA based next generation sequencing (NGS) platforms such as those developed by Illumina, Agilent or Qiagen. However, other methodologies have recently been employed, including mass array and direct hibrydization techniques such as nCounter and the Nanostring platform, which has the advantages of not depending on enzymatic amplification reactions, being an easy technology, and allowing the analysis of low quantity samples. Although several reports have described the use of nCounter for the detection of ALK, ROS1, and RET fusions in FFPE samples ,7,8, onlMET alterations [METΔex14 splicing and MET very high expression in NSCLC cytological samples and we compare the results with FFPE biopsies.We have previously reported the use of nCounter for simultaneous detection of fusions in ALK, ROS1, and RET ,10 and, erations . In thisSamples were obtained from the Quirón Dexeus University Hospital, Hospital General de Cataluña, Teknon Medical Center and Hospital de Mataro with previous informed patient consent. The study had been approved by the ethical committees of each hospital  and was conducted according to the Declaration of Helsinki. Formalin-fixed, paraffin embedded (FFPE) 4 μm slides, obtained by standard procedures, were stained with hematoxylin and eosin. A pathologist determined the tumor areas and evaluated the percentage of tumor infiltration, which were subsequently macro- or micro-dissected. For nCounter analysis, RNA was extracted with a high purity FFPET RNA isolation kit  according to the manufacturer’s instructions. RNA concentration was estimated using the Qubit 3.0 fluorometer .® and Digital Analyzer® (NanoString Technologies) according to the manufacturer’s instructions. Reporter counts were collected with the nSolver analysis software version 3.0. Samples were considered not evaluable if the geometrical mean (geomean) of counts corresponding to the housekeeping genes was lower than 300. Counts from all probes were normalized in two steps and samples were categorized according to previously described threshold values [nCounter  is a fluorescence-based technique for multiplexed digital transcript profiling without enzyme requirements, amplification, or generation of cDNA. Total RNA (between 5–250 ng) was hybridized with a custom-designed mixture (“codeset”) of biotinylated capture tags and fluorescently labeled reporter probes . The codeset also contained probes for housekeeping genes, positive and negative controls . All prod values .® QIAact Custom DNA UMI Panel , which can detect mutations in ALK, BRAF, CDK4, CDK6, EGFR, ERBB2, ERBB4, FGFR1, IDH1, IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, RICTOR, ROS1, STK11, TP53, and copy number variations (CNVs) in BRAF, CDK4, CDK6, EGFR, FGFR1, ERBB2/HER2, KRAS, MET, RICTOR. For the GeneRead panel, up to 40 ng of purified DNA were used as a template. Clonal amplification was performed on 625 pg of pooled libraries and, following bead enrichment, the GeneReader instrument was used for sequencing. Qiagen Clinical Insight Analyze (QCI-A) software was employed to align the read data and call sequence variants, which were imported into the Qiagen Clinical Insight Interpret (QCI-I) web interface for data interpretation and generation of final custom report.The GeneRead DNA FFPE Kit  was used for DNA extraction from FFPE samples, as described . DNA-basMETΔex14 and EML4-ALK amplification using a 20 µL reaction (45 cycles) and visualized in agarose gels, as described [RNA was retrotranscribed with the M-MLV retrotranscriptase  and random primers. HotStart Taq polymerase (Qiagen) was used for escribed ,11. Posi® SPEC ROS1 Dual Color Break Apart Probe  and ZytoLight® SPEC MET/centromere 7 (MET/CEP7) Dual Color Probe  and, according to manufacturer’s instructions. MET immunostaining was performed with SP44 clone  and ALK with the FDA-approved Ventana anti-ALK rabbit monoclonal primary antibody . In both cases, a BenchMark ULTRA automated tissue staining system was used . Membrane staining was graded as described [FISH for ALK, ROS1 and MET was performed on 4 μm sections using the FDA-approved Vysis LSI ALK Dual Color Break Apart Probe , the ZytoLightescribed ,11.From January 2017 to December 2019, we analyzed 151 samples by nCounter, including FFPE biopsies, cell blocks and cytological smears. Of them, 16/151 (11%) did not meet the quality requirements on nCounter analysis (geomean of housekeeping counts < 300) and were considered non evaluable. Three non-evaluable samples were cell blocks, one was a cytological smear and the remaining 12 were FFPE biospsies .n = 135), 120 corresponded to FFPE biopsies, 14 were cell blocks, and one a cytological smear. All 15 evaluable cytological samples had more than 40% of tumor cells and presented adenocarcinoma histology. After purification, RNA concentration ranged between 1.65–49.3 ng/µL, as quantified by Qubit (n = 14) and biopsies (n = 56)  vs. cytological samples (n = 15) , including ALK fusions (n = 5), METΔex14 transcripts (n = 1) and very high levels of MET mRNA expression (n = 1). In contrast, among the 120 valid biopsy samples, 16 (13%) harbored ALK (n = 7), ROS1 (n = 2), or RET fusions (n = 1), and METΔex14 (n = 3) or very high MET expression (n = 3) by nCounter  and high protein expression (histoscore = 240), as expected , ERBB2 (n = 2) and CDK4 (n = 1) and mutation in ERBB2 (n = 1) were detected, which were mutually exclusive with fusions and MET skipping variant. No mutations in driver genes such as KRAS or EGFR were found.Finally, 11 out of 15 cytological samples yielded valid results by NGS. Amplifications in EGFR  and one partial response .Clinical data were available from four patients in which ALK or MET alterations were detected in cytological samples. One patient with The use of multiplexed panels for the detection of mutations and fusions in the lung cancer clinical environment has been exponentially increasing over the last few years ,13. PrevThe nCounter platform is increasingly being used for fusion detection in NSCLC and other types of tumors ,19,20,21n = 1) and RET (n = 1).In our cohort of 104 FFPE biopsies we found 6% ALK positive samples, 4% of specimens with MET alterations and 3% of RET and ROS1 fusions . These pCell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using a multiplexed RNA-based hybridization technology such as nCounter.


CACNA1A, the gene encoding the pore-forming α1 subunit of P/Q-type voltage-gated Ca2+ channels. Although pharmacological treatments, such as acetazolamide and 4-aminopyridine, exist for EA2, they do not reduce or control the symptoms in all patients. CACNA1A is heavily spliced and some of the identified EA2 mutations are predicted to disrupt selective isoforms of this gene. Modulating splicing of CACNA1A may therefore represent a promising new strategy to develop improved EA2 therapies. Because RNA splicing is dysregulated in many other genetic diseases, several tools, such as antisense oligonucleotides, trans-splicing, and CRISPR-based strategies, have been developed for medical purposes. Here, we review splicing-based strategies used for genetic disorders, including those for Duchenne muscular dystrophy, spinal muscular dystrophy, and frontotemporal dementia with Parkinsonism linked to chromosome 17, and discuss their potential applicability to EA2.Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder characterized by paroxysmal attacks of ataxia, vertigo, and nausea that usually last hours to days. It is caused by loss-of-function mutations in  Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder characterized by recurrent disabling attacks of imbalance, vertigo, nausea and ataxia, typically lasting hours to days ,2. SymptCACNA1A, the gene encoding the pore-forming α1 subunit of P/Q-type voltage-gated Ca2+ channels  [V2.2) and R-type (CaV2.3) VGCCs. P/Q-type channels are however more efficient than the other two types of VGCCs in supporting synaptic transmission at mature synapses, partly because they are more tightly coupled to the neurotransmitter release machinery [CACNA1A gene, located on chromosome 19p13, contains 47 exons, many of which are subject to alternative splicing (AS). As a consequence, the total number of CACNA1A splice isoforms is estimated to be in the order of thousands. Moreover, functional studies indicate that some of them exhibit differential properties and expression patterns [2+ signaling to specific cellular tasks.EA2 is caused by mutations in  CaV2.1) ,7. Theseachinery ,9,10. Thpatterns . This crCACNA1A and result in truncated channels generating little or no current. EA2 mutations are found in all regions of the channel but most of them reside in the pore loop region  and an acceptor site at the 3′ end of the intron defined by a polypyrimidine tract and the actual intron/exon junction. The 3′ splice site is functionally coupled to the branch point, which is usually within 40 nucleotides upstream of the 3′ splice site.The splicing reaction is catalyzed by a large complex, the spliceosome, which is composed of five small nuclear RNAs (snRNAs) and more than 200 proteins . snRNAs cis-regulatory sequences, known as exonic and intronic splicing enhancers/silencers (ESE/S and ISE/S), recruit specific RNA-binding proteins, such as serine/arginine-rich (SR) family proteins and heterogeneous nuclear ribonucleoprotein particles (hnRNPs) [Additional (hnRNPs) . These a(hnRNPs) ,66,67.The most common type of AS is exon skipping, also known as cassette exon, in which a whole exon can be either included in or spliced out of the mRNA. Other types of AS comprise intron retention, in which an intron is included in the mature mRNA, mutually exclusive exons, whereby the transcript contains only one of two (or more) possible exons, and alternative 3′ and 5′ splice site selection, leading to alternate inclusion or exclusion of part of an exon ,69.trans-acting elements, can be pathological. Several therapeutic approaches aiming at correcting aberrant splicing have therefore been developed, as detailed below.Aberrant splicing is associated with a growing number of diseases ,71,72. MThe antisense oligonucleotide (AON) approach is based on short oligonucleotides that bind the pre-mRNA and modify gene expression by either promoting mRNA degradation or interfering with the splicing process ,74,75,76SMN1) gene [SMN1, referred to as SMN2, which differs from SMN1 in only few bases. Interestingly, the severity of SMA is inversely correlated to SMN2 expression levels [SMN2 fails however to fully compensate for the lack of SMN1 because of a C-to-T substitution at position 6 of exon 7, which disrupts an exonic splicing enhancer, thus promoting skipping of this exon with consequent formation of an unstable protein [SMN1, researchers have designed AONs targeting an intronic splicing silencer (ISS) in SMN2 to prevent the binding of negative splicing factors, thus favoring exon 7 inclusion in the SMN2 mRNA. This increases the amount of SMN protein in human fibroblasts and in the mouse brain [SMN2 mRNA, has been delivered in patients using intrathecal injections. These clinical trials indicated that the drug is generally well tolerated and effective in alleviating SMA symptoms and improving motor functions [SMN2 transcripts containing exon 7, as well as an increase in SMN protein, as compared to untreated patients [One disease for which AONs have been used to promote exon inclusion is spinal muscular atrophy SMA; . SMA is N1) gene . It resuN1) gene ,85,86,87n levels ,89. SMN2 protein ,91. Instse brain ,93,94. Functions ,97,98,99patients . Followipatients . Ongoingpatients .SMN-AS1, a long noncoding RNA (lncRNA) arising from the antisense strand of the SMN genes, represses SMN expression. AONs degrading SMN-AS1 increase SMN expression in patient-derived cells and in the mouse brain. When lncRNA-targeting and splice-switching AONs are combined there is an even larger increase in SMN levels containing exon 7 [Interestingly, combining different AONs can have synergic effect. For example, g exon 7 .DMD gene that encode the cytoskeletal protein dystrophin. Patients manifest initial symptoms, such as muscular weakness and walking abnormalities, within the first years of life, and lose the ability to walk by the age of 12–14 years. Children with DMD eventually develop also cardiomyopathies and breathing problems due to weakness in respiratory muscles, which eventually lead to death in late adolescence [DMD gene [AON-based approaches have been used as therapeutic strategy also in Duchenne muscular dystrophy DMD; . This islescence ,104. Amolescence . Most DMDMD gene ,107. By DMD gene .DMD. Two main types of compounds have been investigated: drisapersen, a 2′OMePS, and eteplirsen, a phosphorodiamidate antisense morpholino oligonucleotide (PMO), both of which promote skipping of exon 51 by binding to exon 51-splicing enhancers on the DMD pre-mRNA [Over the last years, researchers have developed AON-mediated approaches that promote exon skipping of specific exons in order to restore the open reading frame of pre-mRNA ,110. Skipre-mRNA ,112. Despre-mRNA ,112, phapre-mRNA ,114. Dripre-mRNA ,114. By pre-mRNA . The extpre-mRNA .Another class of promising drugs for treating DMD are tricyclo-DNAs (tcDNA). These are conformationally constrained AONs specially designed to limit torsional flexibility of the sugar backbone in order to stabilize tcDNA/RNA heteroduplexes . Their eDMD gene have been described in DMD. To circumvent this issue, ongoing studies aim at developing multiple exon skipping strategies [Although deletions flanking the exon 51 are the most common cause of DMD , multiplrategies .In addition to promoting inclusion or skipping of exons, AONs can block the recognition of cryptic splice sites. This approach has for example been used successfully to correct the genetic defects of ataxia-telangiecatasia and Hutchinson-Gilford progeria syndrome .ATM gene, which encodes the ubiquitously expressed serine/threonine kinase ATM, involved in controlling cell cycle checkpoints and in repairing damaged DNA [ATM: (i) 7865C3T (A2622V), which creates a new 5′ splice site within exon 55, resulting in a deletion of the last 64 nt; (ii) 513C3T (Y171Y), which activates a cryptic 3′ splice site within exon 8, inducing deletion of the first 22 nt of exon 8; (iii) IVS28-159 A3G in intron 28, which activates cryptic 5′ and 3′ splice sites, resulting in insertion of a 112 nt segment from intron 28. The AMOs were effective in masking the pathogenic cryptic splice sites, thus restoring correct splicing and increasing functional ATM protein levels [ATM mutations [Ataxia-telangiecatasia is a rare, neurodegenerative, autosomal recessive disease characterized by cerebellar degeneration, telangiectasia (spider veins), immunodeficiency, cancer susceptibility and radiation sensitivity. It is caused by mutations in the aged DNA . About 5aged DNA ,121,122.n levels . Furthern levels . Similarutations ,121, thuLMNA gene, which encodes two nucleophilic A-type lamins, lamin A and lamin C. The most common mutation is a de novo substitution c.1824C>T in exon 11, which has the double effect of creating a cryptic splice site and disrupting a probable exon splicing enhancer [Hutchinson-Gilford progeria syndrome is a rare autosomal genetic disorder whose main feature is premature aging . The disenhancer ,127. Theenhancer . Likewisenhancer .trans-splicing (SMaRT). It consists of a trans-splicing reaction between an exogenous pre-trans-splicing RNA molecule (PTM) and a target endogenous pre-mRNA that induces the formation of a chimeric mRNA [A different approach for reprogramming RNA splicing is spliceosome-mediated RNA ric mRNA . A typicric mRNA . Dependiric mRNA ,132.Trans-splicing with a PTM containing the coding sequence of exons 10–24 was effective in correcting this mutation at the mRNA level in both cultured human epithelial cells and a xenograft model of the disease [CF is a common autosomal recessive disease due to loss-of-function mutations in a gene for a chloride channel, cystic fibrosis trans-membrane conductance regulator (CFTR). Deficiencies in CTFR cause the production of mucus secretions with higher viscosity, eventually obstructing pancreatic and respiratory tracts . The mos disease ,136.MAPT gene, which encodes the microtubule associated protein tau, and whose mutation causes several pathologies including FTDP-17. MAPT contains 16 exons. AS of exons 2, 3, and 10 generates six different isoforms. In particular, exclusion or inclusion of exon 10 gives rise to isoforms containing three (3R) or four (4R) microtubule-binding repeats, respectively [SMaRT has also been used successfully to regulate AS in the ectively ,138. Norectively ,140,141.ectively ,143,144.trans-splicing [Likewise, a SMaRT strategy has been developed to compensate for the deficiency of functional SMN proteins in SMA ,147,148.splicing  and coulsplicing ,148.In recent years, the CRISPR/Cas9  system has emerged as a powerful tool for developing new gene editing therapies ,150. It SMN2, thus compensating for SMN1 deficiency in SMA Cas9. The chimeric protein can be targeted to specific DNA sequences by gRNAs where it mediates a C to T conversion without inducing a DSB [DMD in iPSCs derived from a DMD patient who lacked dystrophin because of a deletion of exon 51  and CaV2.3 . CACNA1B and CACNA1E are homologous to CACNA1A  and, like CACNA1A, are extensively spliced, with the splicing pattern often conserved across the three mammalian CaV2 channels [V2.1 induces a compensatory up-regulation in the expression of the other two synaptic VGCCs [V2.2 and CaV2.3 channels maintain a loose and inefficient coupling configuration to the neurotransmitter release machinery [V2 genes. Some interesting data indicate however that it is also extensively influenced by alternative splicing. For example, the mutually exclusive exons 37a (EFa) and 37b (EFb) are conserved across CaV2.1, CaV2.2 and CaV2.3 channels [V2.3, the EFa isoforms of both CaV2.1 and CaV2.2 are more efficient than the respective EFb isoforms in supporting synaptic transmission [V2.2 is, however, enriched only in capsaicin-responsive nociceptors of dorsal root ganglia [V2.2 in most brain regions [V2.2 (and possibly of CaV2.3) in the brain could therefore help compensate for a CaV2.1 deficiency. Importantly, such strategies that leverage a generalized up-regulation of ‘secondary’ presynaptic Ca2+ channels (CaV2.2 and CaV2.3) in EA2 rather than targeting individual EA2 mutations in the ‘primary’ presynaptic Ca2+ channel (CaV2.1), of which there are more than 100, would have the advantage of being suitable for most EA2 patients.As for SMA, where therapies target channels . Notablyic VGCCs ,166. Desachinery ,170,171.channels ,172. Whismission ,173,174. ganglia , where i regions . Molecul


The purpose of our study was the clinical characteristics, radiographic appearance, and outcomes after treatment in patients with rapid destructive arthrosis (RDA) due to subchondral insufficiency fracture (SIF) of the shoulder. Twenty-two cases of RDA of the shoulder were retrospectively reviewed. Clinical outcomes for 15 cases who underwent shoulder arthroplasty were evaluated at an average of 41.4 months. The mean age of patients was 73.7 years (range 50–83 years), and there were 20 women and 2 men. The mean time from onset of symptoms to head collapse was 6.8 months (range 1–12 months). The mean t-score of bone mineral density was −3.1. Nine patients had pseudoparalysis. Based on radiographic appearance, a diversity of types of head destruction with subchondral fracture, bone marrow edema, joint effusion, and synovitis were observed in all cases. In conclusion, RDA due to SIF of the shoulder, presenting with severe short-term pain and functional disability, commonly occurred in elderly women with bone fragility. MRI revealed bone marrow edema, extensive joint effusion, and synovitis as well as a diversity of types of head destruction with subchondral fracture within several months from onset of symptoms. Rapid destructive arthrosis (RDA) is the marked destruction of a joint within months after the onset of symptoms . This coVery recently, subchondral insufficiency fractures (SIFs) of the shoulder have been described as a cause of rapid joint destruction ,2,4,5. STo the best of our knowledge, only 25 cases of RDA due to SIF of the shoulder have been reported as case reports or small case series ,5,10,11.The primary aim of this study was to investigate the clinical characteristics and radiographic appearance in patients with RDA due to SIF of the shoulder. The secondary aim was to evaluate clinical outcomes after shoulder arthroplasty in these patients.This study was approved by the institutional review board (IRB No: 202003042), approved on 19 March 2020. This retrospective study included 311 patients who underwent or scheduled shoulder arthroplasty at a single institution between 2012 and 2019. Inclusion criteria were as follows: (1) patients with RDA of the shoulder joint; rapid destruction was defined as >25% erosive osteolysis of the humeral head within 6 months according to Lequesne et al.  or >2 mmAll available demographic and clinical evaluations were assessed, including age, sex, side, body mass index (BMI), occupation, bone mineral density (BMD), history of trauma, history of oral steroid therapy, history of alcohol abuse, duration of symptoms, clinical scores  pain score, American Shoulder and Elbow Surgeons (ASES) score, subjective shoulder value (SSV)), and range of motions (ROMs) .To diagnose RDA due to SIF, plain radiographs, computed tomography (CT), and MRI were performed. MRI was performed using a 1.5 Tesler scanner  with a dedicated shoulder coil. We obtained MR T1/T2-weighted coronal, sagittal, and axial images and T2-weighted fat suppression images in at least one plane. One patient did not undergo MRI because of pacemaker insertion and received ultrasonography instead.Based on plain radiographs, CT, and MRI, we analyzed humeral head involvement, humeral head destruction, bone marrow edema, joint effusion, bone debris and calcification, synovitis, rotator cuff tear, arthritic change, and glenoid involvement. Humeral head involvement was classified as follows based on involvement of the entire surface of the humeral head: grade 1 (1/3 involvement), grade 2 (1/3–2/3 involvement), and grade 3 (>2/3 involvement). Humeral head destruction was classified as follows based on destruction of the height of the humeral head: grade 1 (<1/3 destruction), grade 2 (1/3–2/3 destruction), and grade 3 (>2/3 destruction).To rule out infectious arthritis, inflammatory arthritis, or crystal-induced arthropathy, blood tests including differential white blood cell (WBC) count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and fluid analysis of joint aspiration were checked. Histologic examination for resected specimens of humeral head or soft tissues was also performed.Twenty out of 22 patients underwent operative treatment. Two patients who scheduled shoulder arthroplasty decided on conservative treatment instead because of severe restriction of cardiac function or poor general condition caused by several medical diseases. Sixteen patients underwent primary shoulder arthroplasty including 14 reverse total shoulder arthroplasty (RTSA), 1 anatomic total shoulder arthroplasty (TSA), and 1 hemiarthroplasty (HA). Three patients underwent 2-stage RTSA after open debridement and cement spacer insertion because infectious arthritis or osteomyelitis could not be completely ruled out. Two patients underwent secondary RTSA after failed arthroscopic debridement . A youngp < 0.05.The IBM SPSS ver. 22.0  was used for statistical analysis. To compare the preoperative and final clinical scores and ROMs, we used the Wilcoxon signed rank test. Statistical significance was set at 2 (range 17.8–29.8 kg/m2), and the mean t-score of BMD tests was −3.1 ± 1.0 (range −5.2 to −2.1) with osteoporosis in 14 patients and osteopenia in five. Three patients did not perform BMD test. No patients had a history of trauma, oral steroid therapy, or alcohol abuse (The mean age of the patients was 73.7 ± 8.1 years (range 50–83 years), and there were 20 women and two men. The right shoulder was involved in 18 patients and the left shoulder in four. The mean BMI was 24.7 ± 3.1 kg/mol abuse .n = 13 shoulders), grade 2 (n = 8 shoulders), and grade 1 (n = 1 shoulder). Humeral head destruction was classified as grade 1 (n = 11 shoulders), grade 3 (n = 7 shoulders), and grade 2 (n = 4 shoulders) (The mean time from onset of symptoms to head destruction was 6.8 months (range 1–12 months). MRI revealed subchondral fractures that presented with a low signal intensity band on T1-weighted images and high signal intensity on T2-weighted images. A diversity of types of head destruction with subchondral fracture, bone marrow edema, joint effusion, and synovitis were observed in all cases. Bone debris and calcification was observed in 14 patients (63.6%) and rotator cuff tears were observed in 17 cases (77.3%). Arthritic change in the glenohumeral joint was observed in five patients (22.7%) . Glenoidoulders) .The mean WBC count, neutrophil count, ESR, and CRP were 7395/µL , 66% , 31.1 mm/h , and 0.72 mg/L , respectively. No growth of pathogens or crystals was observed in aspirated joint fluid analysis and intraoperative tissue culture.Intraoperatively, resected humeral heads were collapsed with detached articular cartilage. In cases of severe head destruction, articular surfaces were flat and covered by fibrous tissues. Histologically, slender bone trabeculae were identified, and bone marrow was replaced by fat tissue. There was evidence of partial osteoid formation admixed with multinucleated osteoclasts in the fibrotic background. Palisading osteoclasts around the newly formed bone were noted. No significant inflammatory infiltrate, infarcts, and osteonecrosis were identified .p < 0.001). The mean active forward flexion, abduction, external rotation at side, and internal rotation at the back improved, respectively, from 66.3°, 57.0°, 22.3°, and the 5th lumbar vertebral level preoperatively to 140.3°, 122.0°, 54.0°, and the 2th lumbar vertebral level postoperatively (p < 0.001). Two complications following shoulder arthroplasty occurred, fixation failure (n = 1) and loosening of glenoid (n = 1); both patients were treated by RTSA with glenoid bone graft for preoperative glenoid defects. A patient with glenoid fixation failure underwent resection arthroplasty with poor clinical outcome. A patient with glenoid loosening was observed with serial evaluations.Fifteen patients had follow-up periods longer than 12 months after shoulder arthroplasty. The mean follow-up period was 41.4 months (range 12–117 months). The mean VAS pain score, ASES score, and SSV improved, respectively, from 7.4, 25.0, and 26.7% preoperatively to 1.6, 79.8, and 76% postoperatively  or osteopenia (n = 5), except for the three patients who did not receive a BMD test. It is worth noting that our study included two men  with RDA due to SIF of the shoulder. In previous studies, all reported cases were in women. Taken together, RDA due to SIF of the shoulder commonly occurred in elderly women with bone fragility.Since Tokuya et al.  in 2004 Yoshikawa et al.  reportedMRI plays an important role in the diagnosis of SIF, which is often inconspicuous on initial radiographs . MRI revDifferential diagnoses of RDA due to SIF of the shoulder included osteonecrosis of the humeral head, rheumatoid arthritis, infection, crystal-induced arthritis, hemodialysis-related destructive arthropathy, osteoarthritis, and neuropathic arthropathy. Because SIF is a recently proposed disease entity, RDA due to SIF of the shoulder might be misdiagnosed as idiopathic destructive arthropathy, osteonecrosis of the humeral head, Milwaukee shoulder syndrome, infectious arthritis, or osteomyelitis ,13. One Previous studies reported that histologic findings of RDA due to SIF of the shoulder included fragmented bone trabeculae, osteoid formation around the newly formed bone, increased osteoclast, and fracture callus ,5,10,11.n = 17) or arthroscopic debridement (n = 3) depending on the patient’s age, activity, symptoms, degree of head destruction, and presence or absence of a rotator cuff tear [n = 1) and loosening of glenoid after RTSA (n = 1)) were observed in patients with glenoid defect. Therefore, shoulder arthroplasty for these conditions should be performed before the glenoid defect occurs in an attempt to avoid postoperative complications.Similar to RDA of the hip, RDA of the shoulder may not respond well to conservative treatment. In a review of the literature, all reported cases with RDA due to SIF of the shoulder underwent arthroplasty . Our casuff tear ,19. Howeuff tear  highlighThis study has a few limitations. First, it is a retrospective study. Second, it was difficult to understand the natural course of the disease, because it is not a longitudinal study. Third, we did not elucidate the pathogenesis of SIF of the shoulder as a non-weight-bearing joint. Further prospective studies are needed to elucidate pathogenesis, associated factors, and natural history of this disease. However, it is of note that this is the first study to investigate the clinical characteristics, radiologic appearances, treatments, and outcomes of a large series of RDA due to SIF of the shoulder.In conclusion, RDA due to SIF of the shoulder, presenting with short-term severe pain and functional disability, commonly occurred in elderly women with bone fragility. MRI revealed bone marrow edema, extensive joint effusion, and synovitis as well as a diversity of types of head destruction with subchondral fracture within several months from onset of symptoms.


R2 > 10%) and novel QTLs . Additionally, 51 and 27 QTLs were identified for the first time through CIM and MCIM methods, respectively. Colocalization of QTLs occurred in four major QTL hotspots/clusters, viz., “QTL Hotspot A”, “QTL Hotspot B”, “QTL Hotspot C”, and “QTL Hotspot D” located on Chr06, Chr10, Chr13, and Chr20, respectively. Based on gene annotation, gene ontology (GO) enrichment, and RNA-Seq analysis, 23 genes within four “QTL Hotspots” were predicted as possible candidates, regulating soybean seed size and shape. Network analyses demonstrated that 15 QTLs showed significant additive x environment (AE) effects, and 16 pairs of QTLs showing epistatic effects were also detected. However, except three epistatic QTLs, viz., ZYqSL-13-3, ZYqSL-13-4, and ZYqSW-13-4, all the remaining QTLs depicted no main effects. Hence, the present study is a detailed and comprehensive investigation uncovering the genetic basis of seed size and shape in soybeans. The use of a high-density map identified new genomic regions providing valuable information and could be the primary target for further fine mapping, candidate gene identification, and marker-assisted breeding (MAB).Seed size and shape are important traits determining yield and quality in soybean. However, the genetic mechanism and genes underlying these traits remain largely unexplored. In this regard, this study used two related recombinant inbred line (RIL) populations (ZY and K3N) evaluated in multiple environments to identify main and epistatic-effect quantitative trait loci (QTLs) for six seed size and shape traits in soybean. A total of 88 and 48 QTLs were detected through composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 15 QTLs were common among both methods; two of them were major ( Glycine max L.) is one of the most economically important crops, being a rich source of both edible oil and protein, and can fix atmospheric nitrogen through a symbiotic association with microorganisms in the soil, and are used as a model plant for legume research [Soybean  presently document more than 400 QTLs for seed size and shape, and the majority of them are not confirmed (http://www.soybase.org). The previous studies used mostly low-resolution and low-density molecular markers such as simple sequence repeats (SSRs) that often result in larger confidence intervals and make the use of these QTLs less effective in crop improvement [Quantitative trait loci (QTL) analysis has proved as a powerful technique to elucidate complex trait architecture. Over the past two decades, recent advances in marker technology and statistical methods have allowed the identification of many QTLs related to seed size and shape traits. The USDA Soybean Genome Database , and coefficient of variation (CV%) associated with six soybean seed shape and size traits of two RIL populations (ZY and K3N), along with their parents, evaluated across three different environments, viz., 2012FY, 2012JP, and 2017JP, are presented in Mean, range , standard deviation, skewness, kurtosis, heritability  for all six traits (h2) for both RIL populations in individual, as well as combined, environments was above 60%, indicating high heritability for all studied traits (r2) among the six traits related to seed size and shape for both RIL populations are presented in Combined ANOVA results revealed that variations among the RILs of both populations were highly significant (x traits . The envx traits . Heritabd traits . The corR2) of 4.46–22.64%, mapped on 18 soybean chromosomes in both ZY and K3N populations across three environments, viz., 2012FY, 2012JP, and 2017JP , as well as in both RIL populations (ZY and K3N)  in the ZY population , viz., ZYqSL-6-1, ZYqSL-6-2, ZYqSL-6-3, ZYqSL-9-2, ZYqSL-13-2, ZYqSL-14-1, and ZYqSL-15-1, were also identified in a single environment in the ZY population  . Additiopulation . Moreoved 2017JP . Four mapulation . The rempulation .ZYqSW-13-1 was detected in two individual environments, viz., 2012JP and 2017JP, in ZY population and expressed an average of 7.45% of PV  and minor (ZYqST-13-3) QTLs were consistently detected in two individual environments in the ZY population with an average R2 of 11.32% and 6.36%, respectively , compared to the ZY. Notably, the most prominent QTL with the highest logarithm of odds (LOD) score (10.76) in a 23.31cM region was located on Chr06, named ZYqSW-6-2, expressing 14.45% of PV  that displayed negative additive effects with beneficial alleles from Nannong1138-2  to 26.84% (K3NqSLW-20-1)  in the ZY population and expressed a PV of 16.03% , expressing an average PV of 19.24% in the K3N population , with an average PV of 9.17%  across all three individual environments . A singl20-1K3N) . For SLW20-1K3N) . A majorf 16.03% . Besidespulation . The qSLof 9.17% . The remR2 > 10% .ZYqSLT-10-1 and qSLT-20-1K3N were significant and stable QTLs having R2 > 10%, as well as detected in three and two individual environments, respectively  but were environment-sensitive, detected only in K3N-RIL populations , with the highest LOD value of 9.01 in an individual environment, identified at 53.61 cM position on Chr20 and expressing a PV of 26.84%  to dissect the additive effect QTLs and QTL x E interactions. By using the MCIM method, we identified a total of 48 additive effect QTLs distributed on 15 chromosomes for all six traits related to seed size and shape in both the RIL populations and all three environments, which expressed 1.69 to 29.35% of the PV . MoreoveR2 < 10% .ZYqSL-13-4, ZYqSW-13-3, ZYqST-13-4, and K3NqST-10-1 revealed AE effect at all environments, while seven and four QTLs showed AE effect in two and one specific environments, respectively  effects . Howeverectively . The AE ectively .ZYqSW-1-1 and K3NqSLT-20-1, were detected in common, with R2 > 10%, identified for the first time. Hence, these QTLs were considered as the most stable and novel QTLs that could be utilized potentially for gene cloning and MAB of soybean seed size and shape traits.Lastly, we performed a comparative analysis of QTLs detected by CIM and MCIM approaches. A total of 88 and 48 QTLs were identified by CIM and MCIM, respectively. Among these QTLs, 15 QTLs were common and are detected by both methods in the same physical genomic interval, indicating the reliability and stability of these QTLs. Besides, by comparing the physical genomic regions of QTLs identified in both populations (ZY and K3N) and mapping methods (CIM and MCIM), two QTLs, viz., K3NqSL-2-1 and K3NqSL-2-2, K3NqST-9-1 and K3NqST-12-4, K3NqSLT-2-3 and K3NqSLT-7-1, and K3NqSWT-6-1 and K3NqSWT-8-4, possess both significant AA and AAE interaction effects with PV of 1.71–9.70% and 1.68–12.03% expressed, respectively  [Based on the gene annotations, available literature, and GO enrichment analysis, we predicted 26, 19, 35, and 18 candidate genes from “QTL Hotspot A”, “QTL Hotspot B”, “QTL Hotspot C”, and “QTL Hotspot D,” respectively . These g; two (Glyma10g35360 and Glyma10g36440); six ; and six  genes from “QTL Hotspot A”, “QTL Hotspot B”, “QTL Hotspot C”, and “QTL Hotspot D”, respectively . However, for cross-validation and improving the accuracy of QTL mapping results, we used two different methods for QTL mapping, viz., CIM and MCIM. A total of 88 and 48 QTLs were detected by CIM and MCIM methods, respectively, associated with all six traits related to seed size and shape  and mapping methods (CIM and MCIM), two major and novel QTLs, viz., ZYqSW-1-3 and K3NqSLT-20-3, were characterized commonly in both mapping methods. These above seven unique and stable QTLs significantly represent potential loci for the improvement of seed sizes and shapes in soybeans. Hence, identification of many new and unique QTLs in the present study suggests distinct genetic architecture in the population derived from the diverse Chinese cultivated soybean genotypes and the need to use more germplasm for revealing the complex genetic basis of soybeans. The favorable alleles for seed size and shape traits were contributed by both parents of two RIL populations, viz., ZY and K3N. Therefore, it is critical to note that not only the higher phenotype parent contributes beneficial alleles but also the contribution of favorable alleles by lower phenotype parents cannot be disregarded; similar results are also described earlier [The use of high-density bin-maps assisting in QTL identification with tightly linked markers provided a good foundation for analyzing quantitative traits. However, to reduce environmental errors, RILs were planted in three environments (consisting of different locations and years), and each of the environments was statistically different. Jansen et al.  describend shape . Among t earlier .R2 > 10%). Therefore, our results showed the reliability of QTL mapping. Furthermore, these QTLs can be utilized as principal targets to identify the candidate genes and MAS in future studies.The stability of the QTL is essential for use in a breeding program. In addition to novel stable QTLs identified for both seed size and shape traits, this study also identified 37 and 21 QTLs through the CIM and MCIM methods, which have been previously colocalized in the same physical interval by earlier studies (see references in ZYqSW-1-1 and ZYqST-18-2 (additive effect: 0.14) identified in both CIM and MCIM methods; though, ZYqSLT-6-5 (additive effect: 0.001) was detected only in the MCIM method (ZY only)  . The genvariance , consequvariance . In the d shapes . All 16 Colocalization of QTLs on chromosomes for different traits related to seed size and shape were also observed in this study. This colocalization of QTLs linked to related traits on chromosomes was reported earlier in soybeans and referred to as “QTL cluster/hotspots” . In thisSimilarly, earlier studies have also reported QTLs for seed weight  and seedLn gene has a large effect on the number of seeds per pod and seed size/shape [PP2C-1 (protein phosphatase type-2 C) allele from wild soybean accession ZYD7 were found to contribute toward the increase in seed size/shape [Glyma06g04810, Glyma06g03700, Glyma13g17980, Glyma13g21770, and Glyma20g30100 have functions that are related to seed development, ovule development, endosperm, and embryo development, which have been reported to directly contribute to seed sizes and shapes in crop plants, including soybeans [Glyma06g02390, Glyma06g06160, Glyma06g07200, and Glyma13g18730 encode RING/U-box superfamily proteins/protein ubiquitination. The ubiquitin pathway has recently been known to play an essential part in seed size determination [Arabidopsis and rice [Glyma06g08290, Glyma20g28460, Glyma20g28640, Glyma20g29750, Glyma20g28550, Glyma13g22790, Glyma20g29750, and Glyma20g27300, function in lipid storage, seed maturation, and cell growth, which have formerly been reported to determine seed size and shape in oilseeds, including soybeans [GmMYB73 promotes lipid accumulation in soybean seeds, which leads to increased seed sizes in soybeans [Glyma06g09650, Glyma10g35360, Glyma10g36440, Glyma13g17750, and Glyma13g21700, are involved in auxin biosynthesis, responses to auxin stimulus, and responses to ethylene stimulus. The auxin regulates seed weights and sizes in Arabidopsis [Glyma06g10700 functions to regulate the brassinosteroid stimulus, which positively governs seed size [Identification of candidate genes underlying the QTL region is of great interest for practical plant breeding. Earlier studies based on QTL mapping of seed size and shape did not practice mining for candidate genes ,54, and,ze/shape , and, reze/shape . Based oze/shape ,60,61. Asoybeans ,63. Likesoybeans . Genes, bidopsis . Glyma06eed size . Hence, 6:9–F6:11 generations of both RIL populations were planted in three different environments, viz., Jiangpu Experimental Station, Nanjing, Jiangsu Province  in 2012 and 2017 (2012JP and 2017JP) and Fengyang Experimental Station, Chuzhou, Anhui Province  in 2012 (2012FY). Both RIL populations, along with their parents, were planted in a single-line plot of 1 m in length and 0.5 m in width in a randomized complete block design with three replications. In each environment, standard cultural and agronomic practices were trailed, as previously described [In the present study, two related RIL populations, viz., ZY and K3N, consisting of 236 and 91 lines, respectively, were used for elucidating the genetic basis of seed shapes and sizes in soybeans. The ZY and K3N populations were derived through a single seed descent (SSD) method by crossing a common higher seed size parent Nannong1138-2 (N) with two cultivated soybean varieties, viz., Zhengxiaodou (Z) and KeFeng35 (K3), having smaller seed sizes . All theescribed ,69.For the phenotypic assessment of seed size and shape, we collected seeds from the randomly selected ten plants harvested from the middle of each block across three different environments  in both RIL populations. The seed size traits include seed length (SL), seed width (SW), and seed thickness (ST), whereas seed shape was assessed using three different ratios, viz., seed length/seed width (SLW), seed length/seed thickness (SLT), and seed width/seed thickness (SWT). The SL, SW, and ST were measured in millimeters (mm) using the vernier caliper instrument, according to Kaushik et al. . Howeverhttp://www.spss.com) [r) among traits was calculated from the average data using PROC CORR in combined environments. The broad-sense heritability (h2) in RIL populations was estimated using the following equation:n is the number of environments, and r is the number of replications within an environment [Descriptive statistics, such as mean, range , coefficient of variation (CV%), skewness, and kurtosis for above seed size and shape traits in both RIL populations, including their parents, were calculated using the SPSS17.0 software (pss.com) . For eacironment .Genetic map construction began with the extraction of DNA from the young and fresh leaves of both RIL populations, along with their parents, by following the protocol of Zhang et al. . DNA libP = 0.05 to determine whether the QTL was significantly associated with the traits [For QTL analysis, we used the WinQTLCart 2.5 software  and QTLNe traits . The mode traits . The phye traits .ZYqSW-1-1, q indicates QTL, SW stands for the trait (seed width), -1 show the chromosome on which the QTL detected, -1 also indicates the order of QTL identified on the chromosome for each trait, andZY represents the ZY-RIL population in which QTL was detected.QTLs were named by following standard nomenclature , with miR2 > 10% were considered as significant and stable QTLs [http://phytozome.jgi.doe.gov) and SoyBase (http://www.soybase.org), we downloaded all the genomic data within the physical interval position of the major “QTL hotspots”, and candidate genes were predicted based on the gene annotations (http://www.soybase.org and https://phytozome.jgi.doe.gov), as well as previously published literature. Gene ontology (GO) information was derived from SoyBase through online resources: GeneMania (http://genemania.org/); Gramene (http://archive.gramene.org/db/ontology); the Kyoto Encyclopedia of Genes and Genomes website ; and the National Centre for Biotechnology Information (NCBI: https://www.ncbi.nlm.nih.gov). These were used to screen the predicted candidate genes further. Gene ontology (GO) enrichment analysis was conducted for all the genes within the four major “QTL hotspots”, viz., “QTL Hotspot A”, “QTL Hotspot B”, “QTL Hotspot C”, and “QTL Hotspot D”, using agriGO V2.0 (http://systemsbiology.cau.edu.cn/agri-GOv2/) [QTLs identified in two or more than two environments with ble QTLs . By utili-GOv2/) . The frei-GOv2/) .R2 > 10%) and novel QTLs, viz., ZYqSW-1-1 and K3NqSLT-20-1. Besides, 51 and 27 QTLs, identified through CIM and MCIM, respectively, were reported for the first time. All identified QTLs were clustered into four major “QTL cluster/hotspots” and represent the major and stable genomic regions governing the inheritance of soybean seed sizes and shapes. Hence, these “QTL hotspot” regions could be of significant consideration for future soybean breeding. Our study predicted 23 genes as the possible candidates, regulating seed sizes and shapes within the genomic region of four “QTL hotspots”; however, they need further functional validation to clarify their actual roles in seed development. Moreover, our results showed that 15 QTLs exhibited significant AE effects, and 16 pairs of QTLs possessed an epistatic effect. However, except for three QTLs, viz., ZYqSL-13-3, ZYqSL-13-4, and ZYqSW-13-4, all the remaining epistatic QTLs showed no main effects. Hence, the hotspot regions and novel significant stable QTLs identified in the present study will be the main focus of soybean breeders for fine mapping, gene cloning, and the MAB of soybean varieties with improved seed quality and yield.In conclusion, the present study is a detailed investigation for elucidating the genetic architecture of seed sizes and shapes in soybean. In aggregate, 88 and 48 QTLs were detected through CIM and MCIM, respectively, including 15 common QTLs, with two major (