Last but not least, the functions and pathways of XRCCs in HCC were also predicted by cBioPortal and DVAID. The transcriptional levels of all XRCCs in HCC tissues were notably elevated compared with normal liver tissues. Meanwhile, upregulated XRCCs expression was positively associated with clinical stages and tumor grades of HCC patients. Survival analysis using the GEPIA database revealed that high transcription levels of XRCC2/3/4/5/6 were associated with lower overall survival (OS) and high transcription levels of XRCC1/2/3/6 were correlated with poor disease-free survival (DFS) in HCC patients. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated the possible mechanisms of XRCCs and their associated genes participating in the oncogenesis of HCC. Our findings systematically elucidate the expression profiles and distinct prognostic values of XRCCs in HCC, which might provide promising therapeutic targets and novel prognostic biomarkers for HCC patients.The X-ray repair cross-complementing (XRCC) gene family has been revealed to participate in the carcinogenesis and development of numerous cancers. However, the expression profiles and prognostic values of XRCCs (XRCC1-6) in hepatocellular carcinoma (HCC) have not been explored up to now. The transcriptional levels of XRCCs in primary HCC tissues were analyzed by UALCAN and GEPIA. The relationship between XRCCs expression and HCC clinical characteristics was evaluated using UALCAN. Moreover, the prognostic values of XRCCs expression and mutations in HCC patients were investigated  Hepatocellular carcinoma (HCC) is one of the most fatal malignant tumors of the digestive system worldwide, which may cause 31,780 cancer-related deaths in the United States in 2019 according to the prediction by the American Cancer Society , it is sThe X-ray repair cross-complementing (XRCC) gene family including numerous members  mainly participates in homologous recombination to maintain chromosome stability and repair DNA damages. Several members have been reported to be involved in specific diseases. For example, FANCG plays a key role in the occurrence of Fanconi anemia . InheritWith the successful implementation of numerous large-scale sequencing projects, including the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx), biomedical studies have been entering the field of “big data” –10. Overhttp://ualcan.path.uab.edu/) is an open-access platform based on level 3 RNA-seq and pathological files from the TCGA database [UALCAN (database . It can http://gepia.cancer-pku.cn/) is an interactive web server developed recently for analyzing the RNA-sequencing expression data as well as the association between gene expression and prognosis from the TCGA and the GTEx database [P value were calculated and displayed online.Gene Expression Profiling Interactive Analysis  is a user-friendly, interactive website and provides visualization, analysis, and download of large-scale cancer genomics datasets [cBioPortal  [The Database for Annotation, Visualization and Integrated Discovery   was emplt-test. Kaplan–Meier survival plots were generated online with survival curves compared by log-rank test. For all analyses, differences were considered statistically significant if P values were less than 0.05.All statistical analyses were performed on the bioinformatics database online. The differential mRNA expression of XRCCs in HCC tissues was analyzed by Student's In order to assess the precise expression profiles of XRCCs in HCC samples, the differential transcriptional levels of XRCCs between HCC and normal liver tissues were evaluated using the UALCAN database. As shown in After high expression of XRCCs was confirmed in HCC, we speculated that overexpression of XRCCs may correlate with the advanced clinical characteristics of HCC patients. So, we next analyzed the association between mRNA expression of XRCCs with clinical characteristics of HCC patients by UALCAN, including patients' clinical stages and tumor grades. As shown in Furthermore, we used the GEPIA website to evaluate the prognostic values of XRCCs. As shown in We next analyzed the associations between XRCCs mRNA expression and disease-free survival (DFS) of HCC patients, and the results exhibited that high mRNA expression of XRCC1/2/3/6 was significantly associated with shorter DFS of HCC patients Figures , while mNext, we analyzed genetic alterations in XRCCs and their associations with OS and DFS of HCC patients. As was shown in All the results suggested that XRCCs may play the roles of significant oncogenes in HCC. Next, to explore the potential mechanisms that XRCCs participate in the carcinogenesis of HCC, we used cBioPortal to construct a network for XRCCs, and the results revealed that a total of 36 genes were significantly associated with XRCCs alterations . MoreoveAbnormality of cancer genetics is an intrinsic factor in tumorigenesis and has been found to participate in the development and progression of HCC . ExternaIn this research, the transcriptional expressions, genetic alterations, and prognostic values of XRCCs in HCC were analyzed. Our results exhibited that the upregulation of mRNA levels was found in all six XRCCs, and mRNA expression of XRCCs was remarkably correlated with patients' clinical stages and tumor grades in HCC patients. Besides, high transcription levels of XRCC2/3/4/5/6 were associated with lower OS and high transcription levels of XRCC1/2/3/6 were correlated with poor DFS in HCC patients. Moreover, mutations in XRCCs were not frequent, but genetic alterations were significantly associated with poor prognosis. Finally, Kaplan–Meier analysis exhibited that patients with genetic alterations of XRCCs genes had worse OS and DFS in HCC patients. All these findings suggested that XRCCs were essential for HCC oncogenesis and development.As an effective analysis method, GO and KEGG analyses provide a comprehensive set of functional annotation approaches for investigators to understand the biological meaning behind the list of genes . In our Among the XRCCs, XRCC1 is the most studied in cancerous disease. Overexpression of XRCC1 contributes to the development of ovarian cancer and its high expression was associated with advanced malignancy and poor clinical outcomes in ovarian cancer patients . BesidesXRCC2 had been shown to participate in chemoresistance to 5-Fluorouracil in colorectal cancer . An incrSimilar to XRCC1 and XRCC2, XRCC3 has been researched widely in the correlation between gene polymorphisms and the risk of cancer. Several studies have revealed multiple gene mutations, including rs861539 C > T, rs1799796 A > G, C241T, which are significantly associated with enhanced risk of HCC , 39. BesXRCC4, a member of XRCCs, had been reported to be an independent prognostic factor for HCC, and high XRCC4 expression was remarkably associated with HCC pathological features . Geneticvia the Wnt/β-catenin signaling pathway [XRCC5 was found to promote the development of numerous cancers, including gastric cancer and colon cancer , 46. Rec pathway . A liter pathway . Besides pathway .Noticeably, there were some limitations to the current study. First, all the data involved in our study was obtained from online websites. Although the field of large data is the frontier of biomedical research, some unavoidable problems must be taken seriously. Mostly, sequencing databases, including TCGA and GTEx, only provide gene expression data at the mRNA level, which may not fully represent the expression even the activation of the candidate genes in the protein level. Further studies should apply western blotting as well as other protein detection techniques to validate our findings in protein level and explore the potential mechanisms of distinct XRCCs in HCC. Besides, further exploration of the clinical application of the XRCCs members in the targeted therapy of HCC should also be conducted in the future.In summary, we systemically analyzed the expression profiles and prognostic values of XRCCs in HCC. Our results revealed that the overexpression of six XRCCs was found to be remarkably associated with clinical stages and tumor grades in HCC patients. Besides, higher mRNA expressions of XRCC2/3/4/5/6 were found to be significantly correlated with OS in HCC patients, while higher mRNA expressions of XRCC1/2/3/6 were notably correlated with favorable DFS. Overall, our research provided a systematic insight into the heterogeneous and complex roles of XRCCs in the carcinogenesis of HCC.


Liver diseases are one of the leading causes of mortality in the world, mainly caused by different etiological agents, alcohol consumption, viruses, drug intoxication, and malnutrition. The maturation of gene therapy has heralded new avenues for developing effective interventions for these diseases. Derived from a remarkable microbial defense system, clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 system (CRISPR/Cas9 system) is driving innovative applications from basic biology to biotechnology and medicine. Recently, the mutagenic function of CRISPR/Cas9 system has been widely adopted for genome and disease research. In this review, we describe the development and applications of CRISPR/Cas9 system on liver diseases for research or translational applications, while highlighting challenges as well as future avenues for innovation. In 2018, a significant breakthrough in gene therapy was achieve by Professor Zhang F and Professor David Liu, showing that single-base editing technology can accurately edit individual bases on DNA or RNA with a very high degree of flexibility and efficiency The CRISPR/Cas9 has been particularly hot in the field of liver research. For example, the CRISPR/Cas9 system was capable of disrupting the intrahepatic HBV genome, with a significant increase of hepatitis B surface antigen (HBsAg) in an HBV hydrodynamics-mouse model Figure 3).Recently, the CRISPR/Cas9 system is utilized as a precise and time- and cost-saving technique for gene KO, while liver diseases vary and suitable options for the therapy adopting CRISPR/Cas9 technique are often limited Hepatitis B virus (HBV) infection is a major global health problem, despite the availability of effective vaccines in vivo models, which will eliminate the virus production efficiently in vivoLin et al. were the first group to use CRISPR/Cas9 to target HBV Table 1).HCV (hepatitis C virus) is one of the leading causes of chronic hepatitis, liver cirrhosis and HCC and infects approximately 170 million people worldwide in vivo and are capable of eradicating multiple cccDNA copies present in infected hepatocytes Additional studies are needed to assess whether CRISPR/Cas9 holds the therapeutic potential to limit productive infections HCC is the fifth most common tumor and the third leading cause of cancer mortality worldwide in vitro and inhibited HCC tumorigenicity in vivo, suggesting that targeted applications of CRISPR/Cas9 system could disrupt the HCC development both in vitro and in vivoin vitro and in vivoTable 2).G9a is a lysine methyltransferase and its primary function is to di-methylate lysine 9 of histone H3 (H3K9me2). G9a-dependent H3K9me2 can recruit H3K9me2-binding proteins preventing transcriptional activation, which leads to gene silencing Most recently, by using CRISPR/Cas9-based functional genomics screening of the human kinome , researchers have found that a set of cyclin-dependent kinases (CDKs), which regulates gene transcription, may be a potential therapeutic target for HCC. Further studies have also indicated that there may be a subgroup of HCC cells with a profound dependence on CDK7 for survival and CDK7 may represent a novel therapeutic target in this subgroup. The high-throughput CRISPR/Cas9 screening technique was first applied to explore the treatment strategy of HCC, providing new approach towards the prevention and treatment of HCC Metabolic liver diseases are better candidates for genome editing correction because many of them are typically severe, refractory to drug therapy and require orthotopic liver transplantation. For some, a low level of gene correction could significantly improve the disease phenotype The potential to correct a Fah mutation mediated by CRISPR/Cas9 has been demonstrated in hepatocytes in a mouse model of HTI. They injected the delivery of Cas9, sgRNA and a coinjected single-stranded DNA(ssDNA) containing the wild-type G nucleotide and homology arms flanking the sgRNA target region by non-viral hydrodynamic injection in the mouse model, which resulted in initial genetic correction rate of ~1/250 cells. The expansion of Fah-positive hepatocytes is sufficient to restore the weight loss of a mouse model of HTI in vivo, hepatacytes has been converted from tyrosinaemia type I into the benign tyrosinaemia type III. Then edited hepatocytes will replace the entire liver in only a few weeks. Hpd excision can change tyrosine catabolism, preventing the accumulation of tyrosine and toxic catabolites. Compared with gene therapy, metabolic pathway reprogramming doesn't need to express the wild-type protein of the disease-causing gene continuously and the protein may cause an immune response, limiting long-term expression A new method called metabolic pathway reprogramming was used to cure HTI mice model successfully. Hydroxyphenylpyruvate dioxygenase (Hpd) is the enzyme that catalyzes the second step of tyrosine catabolism. By deleting Hpd with CRISPR/Cas9 It has brought up new ideas about treating HTI and the approach may be suitable for the treatment of a range of metabolic liver diseases. Because of its flexibility and ease in adjusting the target site, CRISPR/Cas9 system has many possibilities in the therapy of metabolic liver diseases.Recently, novel designer enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, have provided technologies for developing advanced therapeutic strategies Figure 4).Researchers attempted to explore the mechanisms of the tumorigenicity suppression conferred by CRISPR/Cas9-mediated disruption of HBsAg in HCC cells. The results have demonstrated that HBsAg-KO in HCC cells can decrease interleukin (IL)-6 production and inhibite signal transducer and activator of transcription 3 (STAT3) signaling, which highlighted the tumorigenic role of HBsAg. It suggests that the IL-6/STAT3 pathway may be implicated in the HBsAg-mediated malignant potential of HBV-associated HCC The treatment of liver diseases has been sophisticated, which requires and calls for new and better technologies. The ability to edit specific DNA sequences is of paramount importance to advance gene therapy for application to liver diseases The greatest advantage of the CRISPR/Cas9 system is its simplicity and wide applicability in genome manipulations of almost all biological systems tested to date, including cell lines, stem cells, yeasts, worms, insects, rodents, and mammals. Researchers can also mutate amino acids of Cas9 that are critical to DNA catalysis to produce dead Cas9 (dCas9), which still binds DNA but lacks endonuclease activity. When targeting the transcription start site (TSS) of genes, dCas9 can physically block the passage of RNA polymerase, leading to gene silencing To the best of our knowledge, the most exciting application of the CRISPR/Cas9 technology is its potential in the treatment of human diseases. There have been a growing number of preclinical trials targeting various human diseases even though it is currently still at its early stage for clinical applications in vivoOne of the foremost challenges is the off-target activity The second obstacle is the PAM restriction. The precision of the CRISPR/Cas9 DSB activity requires the presence of PAM. CRISPR/Cas9s from different bacterial species have been found to have different PAMs with various lengths and nucleotide compositions. Each type of PAM determines the cutting frequencies of the CRISPR/Cas9 for a given genome. None of the PAMs identified so far or even a combination of all of the known PAMs could cover any whole genome sequences, which may restrict the use of CRISPR/Cas9 technology in some cases. The ideal strategy would be to customize a PAM sequence tailored only to the desired target DNA site through alteration of the responsible PAM interacting amino acids, which has been proved possible by the identification of the amino acids responsible for PAM recognition in both SpCas9 and Staphylococcus aureus Cas9 (SaCas9) The third difficulty is the variations of efficiency. The efficiency of CRISPR/Cas9 directed DSB activities varies widely depending on the nucleotide compositions and genomic context of the target protospacer DNA sites as well as the sgRNA secondary structure. Additionally, the genome editing efficiency is also affected by non-homologous end joining (NHEJ) and homology-directed repair (HDR) DNA repair mechanisms Additionally, there are also some limitations such as the specific delivery to intended target cells and the limitation of immunogenicity of gene modifiers and its delivery agent Initially, CRISPR/Cas9, the bacterial defense system, had been used by microbiologists to understand bacterial immunity. Over the past five years, however, researchers have turned CRISPR/Cas9 into a powerful tool for biological research. The CRISPR/Cas9 genome editing system has accelerated scientific research. With further attempting to combine CRISPR/Cas9 system and the latest technology such as single-cell sequencing and single base editing, the treatment of liver diseases will be upgraded to a new level.As a powerful genome-editing tool, the CRISPR/Cas9 system has been quickly developed into a large-scale function-based screening strategy in mammalian cells in vivo DNA delivery are overcome .Recent works in mammalian synthetic biology have increased the applicability of synthetic logic circuits for human cancer therapy. In our previous study, we have constructed the modular AND gate circuits based on CRISPR/Cas9 system and validated novel genetic circuits that selectively and robustly mediate gene expression in human cancer cells, providing a safe, controllable and specific intelligent gene-editing system Currently, the safest and most effective biological vector for DNA delivery is AAV. In recent years, AAV-based gene therapy drugs have been approved by the US FDA for pre-clinical trials. The All-in-one AAV-Cas9 system recently developed by our group can effectively regulate lipid metabolism in the liver of mice by targeting the Apao1 gene To sum up, this review has discussed CRISPR/Cas9 and highlighted its advantages and disadvantages for applications to the treatment of liver diseases. Several studies have already demonstrated that these technologies may be used in a tissue- and patient-specific manner to disable, augment and correct gene function. Ultimately gene modifiers and cell-based therapies will increase the number of disorders that may be permanently corrected and alter how inborn liver diseases and viral hepatic infections are treated. These technologies present a novel, versatile tool for the treatment and cure of many hepatic illnesses. In the future, we even hope to achieve "rehabilitation" of cells, which is the transformation of tumor cells into normal cells through CRISPR/Cas9, and it will be a technology sweeping the world. When humans begin to modify their own source code of life, we know that a new era has come.Liver diseases are one of the leading causes of mortality in the world and there is an urgent need for effective and safe treatment.CRISPR/Cas9 system has driven progress, improved our understanding of liver diseases and has the potential to revolutionize clinical treatment.The applications of CRISPR/Cas9 systems are rapidly expanding and limitations are being overcome.CRISPR/Cas9 can be readily applied to make basic science discoveries, and also offers exciting prospects for the treatment of liver diseases.


Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable  KCNQ1 (LQTS1), KCNH2 (LQTS2), and SCN5A (LQTS3) being the most common LQTS genes, accounting for ∼90% of all genotype-positive cases (Long QT syndrome (LQTS) is characterized by prolongation of the heart rate-corrected QT interval (QTc) and dysmorphic T-waves on surface electrocardiogram (ECG) recordings, and increases risk of cardiac arrhythmia . QTc prove cases . Alteredve cases . A prolove cases .KCNH2 gene that cause channel dysfunction (loss of trafficking or gating changes that reduce the time channels spend in the open state) are linked to LQTS2, which accounts for ∼40% of LQTS cases , which is responsible for the major repolarizing current, IKr, during phase 3 of the cardiac action potential , and over 72 h, develops nodal activity, separation of atrium and ventricle, and coordinated inter-chamber conduction . This raslow mo zebrafish variant, which resulted in attenuated pacemaking in isolated cardiac myocytes (Isl1) previously identified in mammalian SA nodal progenitor cells, the zebrafish SA node was identified as a ring of tissue at the junction of the sinus venosus and atrium  is a heterogeneous cluster of cells that forms the pacemaker region, responsible for the initiation of cardiac depolarization, and chronotropic responses of the heart. In zebrafish the SA node has been shown to develop very early, with the more primitive “heart tube” showing a constant and linear conduction pathway at 24 hpf. Pacemaking activity was shown to be critical in zebrafish heart rate regulation by the homozygous myocytes . Using ad atrium . Isolatid atrium . Subseqush heart . These ssh heart .At approximately 2 days post-fertilization (dpf), a canal of tissue separates the zebrafish atrium and ventricle. This tissue appears to function similarly to the mammalian atrioventricular node (A-V node), in that it delays electrical propagation between the atrium and ventricle, allowing for coordinated contraction of the two chambers . Using oXenopus oocytes demonstrated functional gap junction formation with similar conductance and voltage-dependence to mammalian Cx40 (s226dco) of zfCx48.5  produced uncoordinated contractions and decreased cardiac output  channels have subsequently been shown to present similar pharmacological sensitivity to blocker compounds, such as terfenadine, as observed in hERG (hKCNH2) channels  or abbreviate  the APD to demonstrate the effect of QT prolonging compounds in zebrafish larvae (breakdance and reggae).Zebrafish were introduced as a drug screening platform in the context of LQTS almost 20 years ago . These schannels . Furtherchannels  as shownh larvae   application increases APD and induces the occurrence of frequent EAD formation (∼1 every s)  . These s90:APD30), which represents prolongation of late phase repolarization or abbreviation of earlier phases, is associated with development of TdP  in zebrafish hearts , which hKCNH2 gene, which encodes the hERG channel. In zebrafish, multiple variants have been identified as possible orthologs to KCNH2, with zkcnh2 initially identified as the primary ortholog. However, screening for all possible zebrafish homologous sequences of zkcnh2, zkcnh6a, which paired more closely as the ortholog to KCNH2 in humans. Tissue-specific RNA extraction and qPCR analyses have confirmed that zkcnh6a is the ortholog of human KCNH2   of zERG channels resulting in a corresponding loss of IKr and 2:1 block when inherited heterozygously, and a silent ventricle phenotype when inherited homozygously . Similar recovery was observed with 9 of 10 non-disease causing SNPs, but not with 39 of 40 LQTS2-associated mutations, from which APD was measured in some and shown to be significantly prolonged (The utility of zebrafish as a model for inherited LQTS2 was more directly demonstrated by the identification of zkcnh6a)  mutationzygously . Recordizygously . These fzygously . Using trolonged . Furtherreggae mutation identified in the zebrafish zERG gene causes a short QT syndrome (SQTS). reggae embryos consistent with the idea that the cardiac phenotype resulted from zERG gain-of-function. Furthermore, crossing of reggae individuals with breakdance individuals (in which repolarization is delayed) produced offspring with no cardiac abnormalities suggesting that the two mutations complemented one another. These studies were pioneering in their demonstration of zebrafish to model inherited LQTS. More recent developments in genetic engineering approaches promise to further unleash the potential of this model species. Below, we discuss future opportunities and challenges using precise gene-editing to create zebrafish models of inherited LQTS.On the other end of QT-related arrhythmia spectrum, the zkcnh6a gene, which provide advanced tools to model and create LQTS in zebrafish. Using transcription activator-like effector nucleases (TALENs) to target double stranded breaks in the genomic DNA immediately after the zkcnh6a start codon, and providing a modified synthetic DNA template for repair, which contained the coding sequence for GFP, these authors first created zebrafish lines expressing the GFP reporter gene in lieu of zkcnh6a. Both homozygous (zkcnh6aGFP/GFP) and heterozygous (zkcnh6aGFP/+) were generated from this approach. zkcnh6aGFP/GFP embryos, homozygous for the GFP knock-in in place of zkcnh6a, i.e., zkcnh6a null, presented contractile defects and pericardial edema consistent with knock-out of zERG function , express GFP exclusively in the heart providing a locus-specific cardiac reporter. Previous fluorescent dye membrane voltage reporters , promise to simplify and improve efficiency of precise gene editing approaches in model systems, including zebrafish . The CRIin vitro Proarrhythmia Assay (CiPA) paradigm advises inclusion of more complex approaches to arrhythmia assessment of compounds beyond heterologous expression of hERG in non-cardiac cells to include computational modeling and iPSC-derived cardiomyocyte platforms to better predict cardiotoxicity. It may be that zebrafish offer a supporting model that could be a cost-effective whole organ early toxicological screening complex model system. Indeed, recent evidence using computational approaches suggest that compound effects in zebrafish readily predict drug efficacy in human cardiomyocytes  to elucidate the phenotype of variants of unknown significance, and to inform clinical risk stratification of patients with hERG variants. Although rapidly evolving, current iPSC-CM approaches are challenged by cellular heterogeneity and immaturity, and they do not yet provide a whole organ or animal model, whereas zebrafish hearts allow for visualization of arrhythmogenicity at the tissue and whole organ level that permits examination of system-wide effects and morphological changes. There is also potential for zebrafish to be used as a whole organ companion model for the development of targeted, patient specific pharmacological approaches. The Comprehensive myocytes .Kr current during the zebrafish action potential. Perhaps as a result of these biophysical differences, the effect of hERG activator drugs is somewhat greater on zERG than hERG channels, and combined with a prominent role for IKr in zebrafish hearts, this could lead to over-estimation of the effects of hERG activator compounds in zebrafish hearts and this needs to be taken into consideration. The zebrafish heart is smaller compared with the human and has reduced ventricular wall thickness, which likely contributes to altered tissue propagation and dispersion of repolarization as has been suggested (Despite these articulated strengths that outline zebrafish as a useful model of LQTS, there are some current limitations/challenges that should be considered and that may influence interpretation or translation of findings to the human. For example, due to ancestral gene duplication events, there are alternate potential gene transcripts responsible for the zERG channel protein, and this requires knowledge and understanding of their interplay in order to carefully design targeted genetic strategies for the generation of specific LQTS-associated mutations. In addition, aside from morphological differences associated with the zebrafish two chambered heart, which might influence interpretations related to regulation of cardiac function, there are some electrophysiological differences in the zebrafish that should be considered. Although similar to hERG, zERG channel biophysical properties show some differences  and thisuggested . With thKS and TC generated tables for the manuscript. KS, RV, ZP, SF, and TC contributed and drafted the manuscript together. GT provided initial input and approved the final draft. All authors approved the final draft of the manuscript.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.


Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root widening and aneurysm if unmanaged. We have previously reported doxycycline, a nonselective matrix metalloproteinases (MMPs) inhibitor, to attenuate aortic root widening and improve aortic contractility and elasticity in MFS mice. We were also first to use multiphoton microscopy, a non-invasive and label-free imaging technique, to quantify and link the aortic ultrastructure to possible changes in the skin dermis. Here, we aimed to assess the effects of long-term doxycycline treatment on the aortic ultrastructure and skin dermis of MFS mice through immunohistochemical evaluation and quantification of elastic and collagen content and morphology using multiphoton microscopy. Our results demonstrate a rescue of aortic elastic fiber fragmentation and disorganization accompanied by a decrease in MMP-2 and MMP-9 expression within the aortic wall in doxycycline-treated MFS mice. At 12 months of age, reduced skin dermal thickness was observed in both MFS and control mice, but only dermal thinning in MFS mice was rescued by doxycycline treatment. MMP-2 and MMP-9 expression was reduced in the skin of doxycycline-treated MFS mice. A decrease in dermal thickness was found to be positively associated with increased aortic root elastin disorganization and wall thickness. Our findings confirm the beneficial effects of doxycycline on ultrastructural properties of aortic root as well as on skin elasticity and structural integrity in MFS mice. With an estimated incidence of 1 in 3,000–5,000 individuals, MFS patients exhibit prominent cardiovascular, skeletal, ocular and pulmonary abnormalities2. FBN1 protein monomers associate with one another to form microfibrils with structural and regulatory functions in the ECM and act as important scaffolds for elastin fiber and collagen deposition, and thus, provide structural integrity3. It is suggested that downstream detrimental effects of FBN1 protein abnormality in MFS is mainly due to disruption of its regulatory role of sequestering the latent form of transforming growth factor beta (TGF- β) in the extracellular space of connective tissue throughout the body2. In the cardiovascular system, degeneration of microfibrils leads to loss of elastin fiber integrity within the blood vessel wall, resulting in aortic elastin fiber fragmentation, disorganization, and reduced load bearing capacity, all of which contribute to aortic root aneurysm, dissections, and rupture as the leading cause of mortality in patients if left untreated4.Marfan Syndrome (MFS), an autosomal dominant genetic disorder affecting the connective tissues, is caused by mutations in the gene encoding the extracellular matrix (ECM) glycoprotein fibrillin-1 (FBN1)6, and that long term treatment with a sub-antibiotic dose of doxycycline, a general and nonspecific inhibitor of MMPs, was effective at reducing MMPs expression levels and improving aortic wall functional and structural parameters, while blocking the progression of aortic aneurysm in a well-established mouse model of MFS8. Furthermore, we have recently demonstrated that the beneficial effects of doxycycline on MFS aortic vessels are associated with significant improvements in pulse-wave velocity (PWV), which is a reliable index of aortic wall stiffness, and aortic wall elasticity in vivo9. However, the long-term effects of doxycycline treatment on aortic elastin and collagen ultrastructure have not yet been fully elucidated. We have previously used multiphoton microscopy (MPM), an advanced label-free imaging technique, to quantify aortic fiber fragmentation and disorganization in the thoracic aorta, as well as the volumetric density of cutaneous elastin in the skin of wild type and MFS mice10, highlighting the notion that structural changes in the aortic wall seemed to be associated with structural changes in the skin dermis of affected mice10. As a follow up study, the present report aimed to use MPM to determine the long-term effects of a low sub-antibiotic dose of doxycycline on aortic elastin and collagen ultrastructure and additionally examine elastic and collagen alterations in the skin dermis of the well-established mouse model of MFS.Using a well-established mouse model of MFS that carries FBN-1 mutation similar to one found in human MFS patients with aortic aneurysm, our group has previously demonstrated that aortic aneurysm progression in MFS is associated with an upregulation of the matrix-degrading enzymes matrix metalloproteinase (MMP)-2 and -9, elastic fiber degeneration, compromised aortic contractility, and endothelial dysfunctionFbn1 allele mutation , the most common class of mutations in MFS10. Heterozygous Fbn1C1041G/+ mice recapitulate all aspects of phenotype observed in MFS human patients, including curvature of the spine, long bone overgrowth, elastic fiber fragmentation within the aortic wall, and progression of aortic root enlargement. Heterozygous Fbn1C1041G/+ mice were bred with C57BL/6 wild-type mice to generate Marfan  and littermate control  mice, which were housed in the institutional animal facility with standard animal room conditions . All animal procedures were approved by the University of British Columbia animal ethics board [reference number A15–0275], and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. . The mouse model of MFS shows signs of aortic elastin fiber fragmentation and an increase in aortic pulse wave velocity  around 3 months of age, which is comparable with 20-year old human MFS patient. In human MFS patients carrying the same fibrillin-1 mutations, the average age for aortic dissection/rupture is around 50-60 years of age that is comparable to 12–15 months of age in mice. Since our study aimed to look at the long-term treatment of doxycycline in MFS adults who are at a higher risk for aortic dissection or rupture, we aimed to run a long-term experiment in mice, and therefore sacrifice mice at 12 months of age. Hence, Beginning at 5–6 weeks of age, mice (n = 4–5) were randomly assigned to CTRL ± doxycycline hyclate  and MFS ± doxycycline hyclate groups for the duration of the study as described previously9.For animal experiments, we used a heterozygous transgenic mouse model harbouring the 2) followed by cervical dislocation, and aortic and skin tissues were collected for various experimental assays.Due to doxycycline instability and sensitivity to light, the doxycycline water was shielded from light and changed every other day throughout the length of treatment. Mice were sacrificed at 12 months of age under inhaled terminal anesthesia . A single transverse section (1–2 mm) was cut from the ascending aortic root above the level of the aortic valve for use in MPM. The transverse aortic root sections were placed vertically on a petri dish and immersed in PBS for MPM imaging. Some aortic sections (2 mm) were embedded in paraffin blocks and sectioned at 5μm thickness for immunohistochemical staining. Furthermore, the skin from the dorsal surface was also dissected out from the same experimental subjects after hairs were removed by very gentle application of hair removal lotion, followed by washing the skin in cold PBS. The harvested skin was flattened out and cut into two pieces. One half was used for MPM imaging and the other was formalin fixed for immunohistochemical staining. Skin samples were also stained for hematoxylin and eosin (H&E) to assess dermal thickness.11. The MPM system consisting of an inverted super resolution microscope (Carl Zeiss LSM 880) with a Ti:Sapphire infrared pulse laser (Coherent Chameleon Ultra) was used to generate two-photon excited fluorescence from elastin and collagen in the samples. With an excitation wavelength of 920 nm, SHG signals originating from collagen were obtained from the emission wavelength of 490 nm. TPF signals originating from elastin were obtained from the emission wavelength range of 400–650 nm with a peak at 500 nm as previously described10.A basic schematic of the MPM system used in this report is illustrated Fig.  and the 12. Elastic fiber fragmentation was determined using Aperio ImageScope software  by tracing individual fibers and measuring their length in pixels followed by unit conversion into micrometers13. A two-dimensional (2-D) Fast Fourier Transform (FFT) algorithm was utilized to determine elastic fiber orientation and anisotropy. Briefly, this technique converts complex spatial patterns of intensity and position from aortic images into directional frequency components using MATLAB software . The details of this technique along with the theory of FFT algorithm are explained in further detail in our previous work10.3-D reconstruction of aortic MPM images, as well as the quantification of volumetric density of elastin and collagen signals was performed using Volocity 6.3 imaging software  as previously described2) was applied to five regularly spaced areas of the aortic ring, and the average positive signal intensity within the multiple regions of interest (ROI = 5) was obtained using the auto-threshold tool. The proportional area of stained tissue for each analyzed section was determined by averaging the five ROIs.Immunohistochemical staining of aortic and skin tissues was performed with the Abcam Mouse and Rabbit Specific HRP/DAB IHC Detection Kit (cat. no. ab236466) per the manufacturer instructions. Briefly, tissue sections were deparaffinized in Histoclear (Fisher Scientific cat. no. 50–899–90147), and rehydrated through a series of descending ethanol solutions. After antigen retrieval in IHC-TEK Epitope Retrieval Solution (cat no. 1W-1100), slides were incubated at 4 °C overnight with the following primary antibodies purchased from Abcam: MMP-2 , and MMP-9 . After washing in 0.5 M Tris buffer, slides were incubated at room temperature for 60 minutes with biotinylated secondary antibody followed by development with DAB solution per the kit instructions. Slides were then dehydrated, cleared, and coverslipped. Negative controls included parallel processing of slides with omission of the primary antibody. Slides were imaged at 200X using a Zeiss Axiostar Microscope. The digitized images were then analyzed using Image J software (NIH). A rectangular sampling tool  was used to compare three or more groups followed by Tukey’s Post-hoc test to correct for multiple comparisons with a P < 0.05 value considered significant.Graph Pad Prism software 8.2 was used for all analyses. Values are expressed as mean ± standard error of the mean (SEM). Comparisons between two groups were made by two-tailed Student’s 10. Here, we assessed the aortic structure in aged 12-months old mice. Representative cross-sectional images of aortic roots taken with MPM are shown to demonstrate the quality of images obtained  receptor type 1 (ATR1) blockers (ARBs) for reducing hemodynamic stress against the aortic wall and in the case of ARBs, to also indirectly downregulate excessive TGF-β signaling18. Unfortunately, clinical data available on β-blockers and ARBs in MFS are scarce and inconsistent, with recent systematic and meta-analyses deeming no benefit of these medications in MFS patients20. However very recently, the AIMS trial has shown an association with a reduction in the rate of aortic dilation in MFS following treatment with the ARB irbesartan21. Despite a reduction in aortic root diameter rates, there is still an apparent overall increase in aortic root diameter over time. Thus, due to the lack of effective available management options, there is a need for more effective alternative management therapies in MFS as either mono- or concomitant therapies.Cardiovascular complications, specifically thoracic aortic aneurysm and dissection, represent the majority of morbidity and mortality factors in MFS23. More recently, we have shown that long-term doxycycline treatment could also correct PWV starting after 18 weeks of treatment, a clinically relevant determinant of changes in aortic wall stiffness and elasticity, which is closely associated with disease progression9. Although previous reports have characterized the anti-remodelling effects of doxycycline treatment on elastic fiber integrity in the aorta of MFS mice, these data were analysed using classical histological techniques involving tissue fixation and staining which can alter native vessel morphology23. This is particularly important in the case of MFS where elastic fiber integrity is already compromised, and where structural analysis mainly involves quantification of the degree of elastic fiber fragmentation or breaks. Furthermore, these studies grade elastic fiber fragmentation and disorganization based on semi-quantitative scoring systems that introduce a wide degree of human error and inter-observer variation. Our group was the first to characterize both elastin and collagen contents in the aorta of MFS mice using MPM10. As a follow up study to our previous report, in the present study, we sought to quantitatively assess the anti-remodelling effects of long-term doxycycline treatment on the aortic ultrastructure of an established MFS mouse model using MPM10. This will compliment our recently reported comprehensive in vivo evaluation of aortic function and structure in live MFS mice subjected to a long-term treatment with doxycycline using ultrasound imaging9.Doxycycline, a nonspecific broad spectrum MMP inhibitor, has been shown by our group and others to be more effective than the current “gold standard” β-blocker atenolol at attenuating MFS-associated aortic widening, and improving elastic fiber integrity and vascular function in the MFS mouse through its inhibition of MMP-2 and -925. In this study, we assessed and determined ultrastructural parameters of the aorta including elastic fiber fragmentation, medial thickening, and total volumetric density of elastin and collagen. Our current data in 12-month old mice showed no significant differences in measurements of total volume of elastin in the aortic root. This is in agreement with our previous observations, where no differences were observed in volumetric densities of elastin in aortic samples from younger  CTRL and MFS mice, suggesting this to be an unsatisfactory parameter for assessing the changes associated with the progression of aortic aneurysm in MFS mice10, and thus, an ineffective measure for assessing the potential beneficial effects of doxycycline treatment. It is important to note that the normal elasticity of the aortic root, which needs to withstand the mechanical stress of pulsatile blood flow, is determined predominantly by the structural integrity of elastic fibers found in the media of arteries, where fragmentation and disorganization of these elastic fibers can lead to dysfunctional blood vessels. Therefore, in this study we have focused on assessing the integrity of elastic fibers in the aortic root using MPM.MPM is a novel and elegant imaging technique that provides several advantages over classical histological methods including specificity, sensitivity, and no requirements for fixation and rough processing of delicate tissues. This is possible due to the nature of elastin and collagen being naturally occurring fluorophores, which can be differentiated by their generation of SHG and TPF signals, respectively10. In this study, using the calculated average length of aortic elastin fibers as a surrogate of elastic fragmentation, we found elastic fibers to be heavily fragmented in the aortic root of aged MFS mice compared to CTRL mice at 12 months of age. Elastin fragmentation was significantly attenuated in MFS mice treated with doxycycline. Interestingly, doxycycline did not influence the degree of aortic medial thickening commonly observed in MFS, suggesting an inherent limitation in efficacy of doxycycline’s anti-remodelling properties in MFS.We have previously shown that elastic fiber fragmentation in the aortic root of MFS mice begins to accelerate at 6 months of age compared to healthy CTRL subjects27, we decided to assess the degree of aortic elastic fiber disorganization using the FFT algorithm as previously described10. In our previous report we showed that at 3, 6, and 9 months of age, the orientation indices (indications of fiber disorganization) for elastin fibers, were significantly reduced as compared to healthy counterparts10. There was also a trend of decreasing orientation index of CTRL aging mice from 3 to 9 months of age10. Our present data shows that at 12 months of age, CTRL and MFS aorta fail to show significant differences in orientation index with approximate values of N = 20–25 for both groups. This lack of difference in the orientation index between CTRL and MFS groups at 12 months of age can be explained by postulating that MFS exhibits accelerated aging processes, which eventually converges with normal aging. Treatment with doxycycline significantly attenuated the decrease in orientation index and elastic disorganization in MFS mice. Interestingly, doxycycline did not exert the same effect on rescuing elastic organization of aging but otherwise healthy CTRL vessel, which may suggest other mechanisms to be involved in breakdown of elastic fibers in natural aging.Since the orientation and organization of elastic fibers can determine important functional properties of blood vessels, including resistance to strain and load bearing strength10. One of the hallmarks of aging involves the decrease of skin thickness, an organ that contains the highest level of elastin and collagen expression in the body. Given doxycycline’s beneficial effects on preserving elastic fiber integrity and organization within the aortic wall, we decided to assess the levels of skin elastin content and dermal thickness in our mice to determine whether inhibition of MMPs expression and activity would have any effect. However, we were unable to detect differences in total elastin content between untreated MFS and CTRL skin samples by MPM, likely because of a high degree of elastin breakdown due to both normal and accelerated aging, given that we have previously shown that at 9 months of age, both CTRL and MFS mice lose a significant amount of their skin elastin content10. As we reported before, the decline in dermal thickness in CTRL mice starts at around 3 months of age (~450 μm) and is dramatically decreased at 9 months of age (~200 μm), which is comparable to levels observed in age- and sex-matched MFS counterparts10. In this study, we show the dermal thickness of both CTRL and MFS groups to be ~200 μm and equal to each other at 12 months of age,. Doxycycline treatment significantly attenuated dermal thinning in MFS-mice. Although not reaching significance, a similar effect on dermis thickness was also observed in 12-month old CTRL mice treated with doxycycline, re-emphasizing the notion that the mechanisms involved in MFS-associated dermal thinning may be similar in some aspects to those of natural aging. To study causation, we measured the expression of MMP-2 and MMP-9, the two MMPs, shown to be increased in the MFS aorta. Despite a trend towards increased MMP expression in untreated MFS versus CTRL skin, we did not observe any significant differences in expression of MMP-2 and MMP-9 in the dermis and epidermis of skin samples collected from untreated CTRL and MFS mice at 12 months of age. Interestingly, doxycycline treatment was able to reduce the expression levels of MMP-2 and MMP-9 in treated MFS mice skin, but not in treated CRTL subjects. This may suggest that signaling pathways other than MMPs are also involved in normal aging of skin in CTRL mice, while MMPs are playing roles that are more prominent in pre-mature and accelerated aging of the skin in MFS mice.We have previously reported that MFS mice show thinning of skin due to a loss of elastin as early as 3 months of ageGiven doxycycline’s robust effect on the attenuation of skin dermal thinning in MFS mice, we assessed if any correlations could be made between several aortic parameters measured in this study versus the degree of dermal thickness in our experimental animals. From the five parameters examined, an increase in total aortic wall thickness, aortic wall elastin thickness, and aortic orientation index were positively correlated with an increase in dermal thickness. However, aortic wall collagen thickness and the length of aortic elastic fibers showed no such association. These positive correlations of dermal thickness to parameters of aortic integrity raise the notion of a potential value of measuring skin dermal thickness in MFS patients as a possible accessory procedure for diagnostic/prognostic purposes. Further careful investigations in the animal models and MFS patients will determine whether the regular and minimally invasive assessment of structural changes in the skin could be of value in determining the state of progression of aortic pathology in these patients.The present study explores the effects of long-term treatment with a sub-antibiotic dose of doxycycline on aortic and dermal ultrastructure in a mouse model of MFS using MPM. We have found MPM to provide higher levels of detail, while reducing histological artifacts related to assessments of aortic elastic integrity in MFS mice. Doxycycline significantly reduces the expression of MMP-2 and MMP-9 accompanied by the attenuation of elastic fiber fragmentation and disorganization in the aorta of MFS mice. In addition, we found that skin dermal thinning observed in MFS is attenuated by doxycycline treatment, and that decreased thickness of the dermal layer is positively associated with increased levels of aortic fiber disorganization and medial thickening. Our findings open a new direction for testing the hypothesis that the degree of skin dermal thickness could possibly be considered as a maker for changes in aortic wall structure and integrity. Further clinical studies that allow for the examination of structural changes in the dermis and epidermis and establishing a potential correlation between dermal skin and aortic events in MFS patients can provide information about the diagnostic or prognostic values of such assessments in these patients.


Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-like receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single-cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic. Mechanisms of endogenous regeneration and repair have been proposed for several mammalian organs . ClassicThe adult kidney is an organ with a low cellular turnover and endowed with progenitors capable of proliferating and differentiating ,5. This Here, we propose an overview of the molecular mechanisms taking place in glomerular and tubular renal progenitors in physiological and pathological conditions  and of hRenal progenitors were discovered by Sagrinati et al. in human kidneys, based on the expression of the stem cell markers CD133 and CD24, in the absence or low expression of differentiation markers ,7. CD133The genetic tagging of Pax2+ progenitors of the Bowman capsule of mice allowed to demonstrate that these progenitors differentiate into podocytes in models of focal segmental glomerulosclerosis (FSGS), and their response to injury determines the outcome of glomerular disorders, further substantiating their role as podocyte progenitors ,12. ReceAn abnormal progenitor response to injury can also contribute to glomerular disorders ,10,14,15Which signaling pathways regulate glomerular progenitor quiescence, proliferation and differentiation toward podocytes in healthy kidneys? Studies on nephrogenesis demonstrated that activation of β-catenin/Wnt signaling represents a pivotal step driving PEC differentiation into podocytes during development ,18. IndeThe control of the cell-fate decision and cell proliferation in many different systems is operated through the integrated signaling of the Wnt and the Notch signaling pathways . LasagniAs the enhancement of renal progenitor differentiation into podocytes may represent an attractive therapeutic strategy to promote the remission of glomerular disorders, several studies have been performed to identify differentiating compounds. Retinoic acids (RA) are derivatives of vitamin A with established benefits in the treatment of a variety of cancers . RA haveEndlich et al. demonstrated the role of Dach1 (Dachshund homolog 1) in the cell fate determination of PEC into podocytes and for proper podocyte function. Podocytes express high levels of Dach1 in vivo and in vitro, while PEC express very low levels of Dach1. The authors found that the induction of Dach1 expression in PEC significantly upregulates the podocyte-specific proteins synaptopodin and WT1. Interestingly, Dach1 is part of the Eya-Six-Hox-Pax regulatory network, and the regulation of synaptopodin expression was accompanied by a concomitant downregulation of Pax2 expression .Guhr et al. analyzed by which mechanisms renal progenitors maintain the potential to express podocyte proteins under pathophysiologic conditions and demonstrated that they contain an activated ubiquitin-proteasome system (UPS) that leads to the rapid degradation of newly synthesized podocyte-specific proteins . On the It is well-known that, in the kidney, the mechanical environment is subjected to modifications in established models of glomerular diseases and can affect the differentiated state of numerous cell types, including podocytes . We receRenin angiotensin aldosterone system inhibitors (RAAS-I) are drugs effective in retarding the progression of kidney disease through a variety of actions. The mechanisms responsible for the therapeutic effects of these drugs, as well as their renal cellular targets, have been largely studied in several animal models of human kidney disease. Recent data demonstrated that they might also exert their beneficial effects by promoting renal progenitor differentiation into podocytes. Indeed, in a rat model of glomerular injury, a treatment with ACE-I induced a reduction of progenitor proliferation, the diminution of crescent formation and avoided the progression toward glomerulosclerosis . Thus, mInjuries to podocytes are considered an important contributor to diabetic kidney disease progression toward end-stage kidney disease ,40,41. SWhile renal progenitors can drive podocyte regeneration following injury , they caCollapsing nephropathy and pseudocrescents also originate from renal progenitors . At diffSeveral recent studies highlighted a critical role for the de novo expression of CD9 and, subsequently, of CD44 as a pathogenic switch of PECs from a quiescent to an activated phenotype in CGN and in FSGS ,51,52, cRenal progenitors from the parietal epithelial layer of the Bowman capsule can potentially regenerate proximal tubular epithelial cells at the glomerulotubular junction . HoweverKumar et al. performed lineage tracing of rare Sox9-expressing cells in the proximal tubule and identified them as a putative tubular progenitor population involved in post-acute kidney injury (AKI) recovery . Sox9 isRecently, Lazzeri et al. provided evidence that tubular progenitors undergo mitosis and replace approximately half of the irreversibly lost tubular cells during AKI . PerformHuman renal progenitors express B lymphoma Mo-MLV (Moloney murine leukemia virus) insertion region 1 (Bmi-1) . Bmi-1 iAnother important molecule involved in the regulation of the tubular progenitor is Toll-like receptor 2 (TLR2), or CD282, an evolutionary conserved membrane protein that plays an important role in pathogen recognition and the activation of innate immunity. TLR2 acts as a sensor of tissue injury through the detection of damage-associated molecular pattern molecules (DAMPs) released by damaged tissues. TLR2 activation leads to the activation of downstream transcription factors that regulate the expression of survival genes or proinflammatory cytokines and chemokines ,76,77. SThe expression of molecules from the Wnt pathway has been reported in adult renal progenitors in mice  and humaAs previously mentioned, Notch signaling is an evolutionary conserved pathway that has a critical role in kidney injury and repair ,91,92,93Several drugs have been shown to improve kidney regeneration, and, among those, histone deacetylase (HDAC) inhibitors (HDACis) may be a promising therapeutic option for the treatment of AKI ,101,102.Biological and molecular features of kidney cancer suggest that renal progenitors could be at the origin of the development of different kidney tumor types.In a recent study, Peired et al. showed that human renal progenitors overexpressing NICD1 had an increased proliferative capacity and form aberrant mitosis in 2D cultures and could generate a tumor-like mass in 3D cultures . SimilarRecently, two studies suggested that angiomyolipomas originate from multipotent kidney epithelial cells localized in the tubule and undergoing clonal expansion in response to tuberous sclerosis complex (TSC) gene deletion ,110. BotWan et al. observed that SOX9 expression was upregulated in RCC patients and correlated with the advanced pathological grade . RCC patThe fast development of scRNAseq is opening new perspectives for dissecting the molecular processes involved in renal progenitor regulation in physiological and pathological conditions. ScRNAseq consists in obtaining gene expression profiling at a single-cell resolution, putting in evidence the different cellular states and molecular dynamics of even the rarer subpopulations. This novel technology has been used successfully in several organs—for example, to study Prominin 1+ liver progenitors , Dach1–dRenal progenitor-based therapies represent a promising new frontier in the treatment of renal diseases, as several studies suggest that they improve kidney function following injury . HoweverMolecules and exosomes secreted by renal progenitors promote recovery from kidney disease through their ability to exert a series of renoprotective and regenerative effects thanks to their reduced immunogenicity and lower risk of maldifferentiation and tumorigenesis compared to cell therapies. These remarkable features make them appealing for clinical applications.Another challenge in the clinical approach to kidney disease is the discovery of new tools for diagnosing and monitoring kidney disease that would be easily accessible with noninvasive procedures. In this context, urine represents a valuable biofluid due to its accessibility, fast and easy sampling and broad variety in proteins, metabolites, cells and cellular contents released from the urogenital tract . The preRecently, it has been proposed that the expression of the renal progenitor marker CD133 in urinary EVs represents a good marker for the evaluation of the functional status of the renal tubular compartment and of the presence of cells with proliferative and repairing activity within tubules after injury. Indeed, two studies reported that CD133+ urinary EV levels, elevated in healthy subjects, not only decrease in patients with acute tubular damage  but, alsUrine-derived kidney cells could also be a powerful personalized tool for functional studies on candidate variants in inherited renal disease. As described by Lazzeri et al., urine-derived renal progenitors obtained from patients carrying pathogenic mutations in genes encoding for podocyte proteins expand in culture but develop anomalies in the expression or localization of podocyte proteins following podocyte differentiation . In agreA vast body of literature describes the numerous mechanisms of the regulation of renal progenitors in the glomerular and in the tubular compartments, allowing us to have a global vision of the complexity of the molecular processes taking place in the physiological and in pathological conditions. Knowing the molecular signature of renal progenitors opens the door to identifying new targets for drugs to sustain kidney regeneration or biomarkers to monitor kidney health.


Human studies support a strong association between hypertriglyceridemia and atherosclerotic cardiovascular disease (CVD). However, whether a causal relationship exists between hypertriglyceridemia and increased CVD risk is still unclear. One plausible explanation for the difficulty establishing a clear causal role for hypertriglyceridemia in CVD risk is that lipolysis products of triglyceride-rich lipoproteins (TRLs), rather than the TRLs themselves, are the likely mediators of increased CVD risk. This hypothesis is supported by studies of rare mutations in humans resulting in impaired clearance of such lipolysis products . Several animal models of hypertriglyceridemia support this hypothesis and have provided additional mechanistic understanding. Mice deficient in lipoprotein lipase (LPL), the major vascular enzyme responsible for TRL lipolysis and generation of RLPs, or its endothelial anchor GPIHBP1, are severely hypertriglyceridemic but develop only minimal atherosclerosis as compared with animal models deficient in apolipoprotein (APO) E, which is required to clear TRLs and RLPs. Likewise, animal models convincingly show that increased clearance of TRLs and RLPs by LPL activation  results in protection from atherosclerosis. Mechanistic studies suggest that RLPs are more atherogenic than large TRLs because they more readily enter the artery wall, and because they are enriched in cholesterol relative to triglycerides, which promotes pro-atherogenic effects in lesional cells. Other mechanistic studies show that hepatic receptors (LDLR and LRP1) and APOE are critical for RLP clearance. Thus, studies in animal models have provided additional mechanistic insight and generally agree with the hypothesis that RLPs derived from TRLs are highly atherogenic whereas hypertriglyceridemia due to accumulation of very large TRLs in plasma is not markedly atherogenic in the absence of TRL lipolysis products. Whether elevated plasma triglycerides play a causal role in exacerbating cardiovascular disease (CVD) or represent a biomarker of CVD risk has been debated for 4 decades . In thisAPOE2 genotype in combination with hormonal and/or environmental factors  and their remnant lipoprotein particles . Chylomicrons are synthesized and secreted from the intestine after a meal, whereas VLDLs are produced by the liver. Chylomicrons contain APOB48, a truncated form of APOB100, as the main apolipoprotein. Elevated TGs or hypertriglyceridemia can be due to greater intestinal and/or hepatic secretion of TRLs or reduced clearance of TRLs from the circulation . TRLs anLPL itself, APOC2, LMF1 (lipase maturation factor 1), GPIHBP1 (glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1), APOA5, and GPD1 (encoding glycerol-3-phosphate dehydrogenase 1) are associated with high TG, although both severe forms of hypertriglyceridemia and moderate-to-mild hyperglycemia are usually of polygenic origin  of cohorts with a range of TGs, and genes identified by studies of families with TG phenotypes, have revealed that genetic factors contribute to a relatively large proportion of variation in plasma TG. In particular, homozygous or compound heterozygous mutations in genes that alter lipoprotein lipase (LPL) activity, like c origin , 11. Bi-reatitis . Furtherreatitis .LPL is the major vascular enzyme catalyzing TG hydrolysis, generating free fatty acids and monoacylglycerol for tissue use, and RLPs. Hence, LPL is the principal player in the clearance of circulating TRLs. The binding of LPL to GPIHBP1 focuses the intravascular hydrolysis of TRLs on the surface of capillary endothelial cells in e.g., adipose tissue, skeletal muscle, and heart . LPL's aAdditionally, receptor-mediated capture of RLPs produced after lipolysis by hepatic LDLR, LRP1 and syndecan 1 contributes to maintaining plasma TG levels .Elevated plasma TGs have been known to associate with an increased risk of atherosclerotic CVD for decades. The first clues emerged over 40 years ago from the finding that patients with familial forms of hypertriglyceridemia exhibited an increased risk of coronary artery disease . Large eWeakening the case for a causal role of hypertriglyceridemia as a CVD risk factor are the clinical trials of TG lowering, which have mostly resulted in a lack of effect on CVD outcomes. Thus, fibrates and omega-3 fatty acids, which lower plasma TGs, have been mostly negative for CVD benefit , althougThus, although the association between hypertriglyceridemia and CVD risk in humans is strong, causal evidence is so far scarce. In fact, subjects with familial chylomicronemia syndrome, which is due to pathogenic mutations in genes controlling LPL itself or LPL activity, do not usually exhibit an elevated risk of atherosclerotic CVD despite the very high levels of TGs , 24. HowThe complex relationship between elevated TGs and atherosclerosis may be explained by a model in which increased atherosclerosis (and CVD) is not caused by the high TG content/lipoprotein particle, but to the presence of RLPs (chylomicron and VLDL lipolysis products), which are smaller than chylomicrons and VLDL and can more effectively exert pro-atherogenic effects in the artery wall . As descThe concept that RLPs are highly atherogenic is supported by the finding that patients with remnant removal disease exhibit an increased risk of premature atherosclerotic CVD , 27. ThiWhile these findings implicate RLPs in promoting atherosclerosis, their exact role in the atherogenic process is still unclear. However, the findings discussed above suggest that lower TG levels might be associated with an increased CVD risk, as compared with severe hypertriglyceridemia, if accompanied by elevated levels of more atherogenic RLPs derived from TRLs.In order to tackle the issue of whether TRLs or RLPs are causatively involved in exacerbating atherosclerosis, animal models have been used to investigate potential atherogenic effects of TRLs and RLPs and to provide mechanistic insight through an approach that has sometimes been termed “reverse translation,” “human-first,” or “bedside-to-bench” research, which begins with observations in the clinic and works “backwards” to uncover the mechanisms behind the clinical observations . In factApoe−/−) mouse exhibits a dramatic atherogenic phenotype concomitant with a lipoprotein profile enriched in VLDL and RLPs even when fed a low-fat diet -cholesterol when analyzed by fast phase liquid chromatography (Ldlr−/− mouse), which was generated as a model of homozygous familial hypercholesterolemia (caused by LDL receptor mutations in humans), on the other hand, exhibits an increase primarily in LDL-cholesterol when fed a chow diet, reflecting impaired clearance of LDL through hepatic LDL receptors  exhibit higher levels of VLDL and chylomicron RLPs than do Ldlr−/− mice, revealed that TRLs and RLPs are also cleared through a receptor (LRP1) distinct from the LDLR  whereas the Ldlr−/− mouse has primarily APOB100 in plasma , which is associated with a dominantly inherited form of remnant removal disease was first described in a large Dutch family  is associated with massive chylomicronemia, low HDL-C levels, and recurrent attacks of pancreatitis when not controlled by a strict diet . In contLpl−/− mice from neonatal lethality  spontaneously develop early fatty streaks when fed a chow diet , suggest content , supportgression . The autgression –91. Thusgression , 91.Ldlr−/− mice as well as Apoe−/− mice overexpressing LPL are resistant to diet-induced atherosclerosis, due to the efficient clearance of TRLs and RLPs in these mice  gene in murine chromosome 17. Subsequent studies showed that LMF1 localizes to the ER membrane and facilitates maturation of LPL, HL, and endothelial lipase (EL) (Combined lipase deficiency (cld) was identified in a spontaneous mouse mutant deficient in both LPL and hepatic lipase (HL) activity . These mase (EL) .LMF1 in adipose tissue, heart, and muscle tissues resulted in increased LPL activity in all of these tissues  is required for the maintenance of normal plasma TG concentrations, and loss-of-function mutations in the CREB3L3 gene are associated with severe hypertriglyceridemia in humans  elevated plasma TG levels have been shown to be due to reduced hepatic TRL clearance through syndecan 1 and flotillin 1-mediated endocytosis (Other mouse models have been used to study the effects of non-genetic conditions that elevate plasma TG levels (referred to as secondary hypertriglyceridemia in humans). For example, mice made diabetic using the beta cell toxin streptozotocin or a virus develop elevated plasma TG levels. Interestingly, in a virally-induced diabetes model (a model of poorly controlled type 1 diabetes), plasma TG levels are elevated without a significant increase in plasma APOB (which is most abundant in LDL), suggesting a selective effect on TRLs . The mecocytosis . Togetheocytosis .The rat was used for some of the early studies on TRL and RLP metabolism. For example, studies in hepatectomized rats demonstrated the importance of the liver in RLP removal  and studThere are also rat models of hypertriglyceridemia. Rats homozygous for the corpulent gene (cp/cp) become obese, insulin resistant, and hypertriglyceridemic, primarily due to hepatic hypersecretion of VLDL . The cp Apoe−/− rats display a range of atherosclerosis phenotypes, ranging from no detectable lesions, to very early signs of lipid deposition and atherosclerosis , developed on the basis of structural insights gained from all-atom molecular dynamics simulations, directly activates LPL and also inhibits APOC3's action. D6PV treatment drastically lowers TGs in APOC2-deficient mice and in human APOC3-transgenic mice . This peprimates . Its effANGPTL3 lead to familial combined hypolipidemia, characterized by low circulating levels of LDL-C, HDL-C, and TGs  mice, in db/db mice and in APOE*3-Leiden mice in which ANGPTL3 is inhibited  resulted in lowered plasma TGs, LDL-C and HDL-C and reduced atherosclerosis  protect against CVD risk, and whether this protection is related to TG lowering or other mechanisms should be assessed in animal models of hypertriglyceridemia. New tools and methods to measure and evaluate TRLs and RLPs will also be critical for understanding if TRLs and RLPs have distinct effects on atherosclerotic lesions and as risk factors for CVD (The multifactorial nature of hypertriglyceridemia makes it challenging to draw clear conclusions concerning its atherogenicity. A more complete understanding of the genes and variants that modulate plasma TGs requires strong functional validation at all stages:  for CVD .The last decade saw the discovery of new drug targets and the early phase clinical trials testing these targets, including APOC3 and ANGPTL3, which both inhibit LPL activity. The development of several different strategies for reducing TG levels is likely to be more specific and effective than drugs currently on the market. Animal models will help to provide mechanistic insight through reverse translation of clinical observations. There is hope that the current decade will reveal whether such strategies will also prevent CVD risk in patients with hypertriglyceridemia and others with impaired TRL and RLP catabolism.DB wrote the manuscript. KB wrote parts of the manuscript and edited the full manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amyloid fibril formation is a hallmark of diverse neurodegenerative and metabolic diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and type 2 diabetes mellitus (T2DM). Conventional diagnosis is based on the appearance of fibrils or plaques, while neglects the role of early-stage oligomers in the disease progression. Recent studies have uncovered that it is the early-stage oligomer, rather than the mature fibril, that greatly contributes cytotoxicity. The formation of oligomers involves complicate structural conversions and it is essential to investigate their conformational changes for a better understanding of aggregation mechanism. The coexistence of soluble early-stage oligomers, intermediates, and pre-fibril species makes it difficult to be differentiate by morphological methods, and only average structural information is provided as they lack the ability of separation. Therefore, mass spectrometry (MS) becomes an alternative technique that presents new and complementary insights into the onset of amyloid fibrils. This review highlights the hotspots and important achievements by MS in the field of amyloid formation mechanism, including the direct detection and differentiation of soluble oligomers (native MS), unambiguous identification of interacted sites involved in the onset of aggregation [hydrogen/deuterium exchange (HDX) and chemical cross-linking (CX)], and conformational switch that leads to fibrilization [collision cross section (CCS) regularity by ion mobility (IM)]. In vitro studies suggest that most amyloid fibril formation follows nucleated polymerization mechanism, involving lag phase and growth phase before mature fibril formed. It has long been recognized that mature fibril is the leading cause of related diseases and techniques are mainly applied for diagnosis of its appearance.Amyloid fibril formation is a hallmark of numerous neurodegenerative and metabolic diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and type 2 diabetes mellitus (T2DM)  and atomic force microscope (AFM), amyloid fibrils ranging from 6 to 130 Å in width and 1,000 to 16,000 Å in length could be detected  in the mass analyzer. Besides measuring the m/z, structural information is provided by tandem MS through dissociation  according to molecular weight measurement. Different conformations of the same oligomer are discovered upon gas-phase separation, which are derived from formation pathways. On the basis, MS has emerged as a highly sensitive technique for amyloid aggregation studies and presents new and complementary insights into the onset of amyloid fibrils, including oligomer characterization, aggregation mechanism investigation, and inhibitor screening. This review surveys the combination of MS with selected analytical techniques and their advanced achievements in aggregation studies (selected studies are listed in Mass spectrometry (MS) is a powerful analytical technique that attracts increasing attention in bioanalytical fields. Molecules are converted to gaseous ions in the ion source and separated according to their mass-to-charge ratios (2). Denaturant, such as organic solvent, is usually added to enhance the MS signal and achieve high detection sensitivity. However, it is not compatible with holding non-covalent bindings within oligomers, and it will cause dissociation during ionization. Therefore, native MS is an alternative ionization method that enables the direct detection of oligomers , infrared multiphoton dissociation (IRMPD), and collision-induced dissociation (CID)  the overlapped species have significant intensity difference; (2) the overlapped species have high charge states so that the isotopic distributions could not be well resolved; or (3) a single oligomer contains different conformations. Therefore, further separation is required.Electrospray ionization (ESI) is one of the soft ionization methods, which has been widely used for protein ionization. During ESI, protein molecules are transferred from solution to the gas phase with the assistance of high voltage (2.5–3.0 kV) and nebulization gas . The gas-phase separation depends on their charges, sizes, and shapes, and leads to drift time difference  showed a doubly charged monomer (denoted a monomer2+) predominantly as well as minor dimer3+ and trimer5+, which were identified according to their m/z and isotope distributions. Upon IMS separation, co-populated species sharing the same m/z were differentiated according to their CCS values, such as dimer3+ overlapped with tetramer6+. Moreover, two CCS values of monomer differed by 15% were resolved, referring to compact and expanded conformers ; σ = αn+κ   to the aggregated form (PrPsc). Their work suggested that PrPsc underwent two-step conversions before fibril formation, including the formation of the β-sheet core in region 159–225, and the structure loss in the α1 region for further monomer addition to pre-fibril. The attachment of monomer to fibril induced two-step conformational changes, including helix 1 unfolding only after helices 2 and 3 transformed into β-sheet . With the advantage of fast sampling timescale (~μs) and native or near-native state detection of proteins, FPOP combined with MS can offer high special resolution structural information of proteins or protein complexes  and AFM helped obtain more information about secondary structure and confirmed that it was a heterogeneous mixture of oligomers that existed during the lag phase  enables direct detection of oligomers  how the mechanism obtained from the simulated system could be verified in real biological systems; and (iii) there are more factors that affect aggregation process during in vivo studies which increases the detection complexity.This review describes the analysis and mechanism studies of amyloid formation by MS coupled with other analytical methodologies, which is complementary to that of biological and morphological methods. Conventional methods offer average structural information by regarding them as an entirety. Early-stage aggregation is actually a mixture of oligomers, which could be clearly differentiated by MS according to their molecular weights. In addition, oligomer ions are further separated in the gas phase by IM so that various conformations are discovered. MS coupling with HDX and CX also enables insight into conformational changes and unambiguous identification of interacted sites involved in the onset of aggregation. However, limitations still exist: (i) current studies are done JH did literature research and created the draft. QZ reviewed and revised the manuscript. All authors read and approved the manuscript.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.


Mallard ducks are a natural host and reservoir of avian Influenza A viruses. While most influenza strains can replicate in mallards, the virus typically does not cause substantial disease in this host. Mallards are often resistant to disease caused by highly pathogenic avian influenza viruses, while the same strains can cause severe infection in humans, chickens, and even other species of ducks, resulting in systemic spread of the virus and even death. The differences in influenza detection and antiviral effectors responsible for limiting damage in the mallards are largely unknown. Domestic mallards have an early and robust innate response to infection that seems to limit replication and clear highly pathogenic strains. The regulation and timing of the response to influenza also seems to circumvent damage done by a prolonged or dysregulated immune response. Rapid initiation of innate immune responses depends on viral recognition by pattern recognition receptors (PRRs) expressed in tissues where the virus replicates. RIG-like receptors (RLRs), Toll-like receptors (TLRs), and Nod-like receptors (NLRs) are all important influenza sensors in mammals during infection. Ducks utilize many of the same PRRs to detect influenza, namely RIG-I, TLR7, and TLR3 and their downstream adaptors. Ducks also express many of the same signal transduction proteins including TBK1, TRIF, and TRAF3. Some antiviral effectors expressed downstream of these signaling pathways inhibit influenza replication in ducks. In this review, we summarize the recent advances in our understanding of influenza recognition and response through duck PRRs and their adaptors. We compare basal tissue expression and regulation of these signaling components in birds, to better understand what contributes to influenza resistance in the duck. Influenza A virus (IAV) is a negative sense single stranded RNA (-ssRNA) virus which causes significant disease in both humans and animals. Due to rapid accumulation of mutations during replication, this virus can change surface proteins quickly, thus escape both natural and vaccine-based immunity. These mutations also affect the pathogenicity of individual viral strains. In chickens especially, IAV can cause severe disease and mortality. The virus is classified as low pathogenic or highly pathogenic avian influenza  depending on the severity of disease that it causes in chickens  surface proteins bind to glycoprotein-linked sialic acid (SA) on the surface of host cells. The specific linkage of these sialic acids allows the virus to not only become specific to different host species, but also different tissues in these hosts. Humans express sialic acid α-2,6 linked galactose  surface molecules on epithelial cells in the upper airways, which is the site of replication for IAV in humans , which also includes the many breeds of domesticated mallard ducks , retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) . TLRs, RBasal expression of these PRRs may also allow different tissues to detect IAV infection earlier. To visualize PRR readiness we show basal expression patterns in different tissues in ducks and chickens . TissuesThe RIG-I like receptor (RLR) family are select cytosolic RNA helicases which contain conserved DExD/H box domains used in nucleic acid binding  can both augment IFN signaling   is involved in antiviral responses and plays analogous roles to that of mammalian IRF3. Recent studies have found that chIRF7 can be induced to translocate across the nucleus downstream of both chMAVS and chicken stimulator of interferon genes (chSTING), and chIRF7 dimerizes following chTBK1 activation, allowing it to increase IFN-β signaling  is a protein on which many PRR pathways converge in order to increase NF-κB and IFN signaling downstream of pathogen pattern recognition . It was Duck STING (DuSTING) shares 43 and 71% identity to human and chicken STING (chSTING), respectively   and signal downstream to produce IFNs and other cytokines through their cytoplasmic Toll/IL-1 receptor (TIR) domain , lipopolysaccharide (LPS) and CpG ODN can reduce IAV replication in both mammals   can form multi protein complex inflammasomes, which possess autocatalytic activity. This activity can activate caspase-1 and induce the production of proinflammatory cytokines IL-1β and IL-18 . NLRP3 iTissue expression of NLRP3 differs between ducks and chickens . NLRP3 iIRF1 is known to be an activator of IFNs though several mechanisms, but one of importance is its regulation of the NLRP3 inflammasome  in response to both poly (I:C) stimulation and infection with a mouse-adapted strain of H1N1  are a family of proteins which have diverse functions in the cell such as mediating apoptosis, sequestering viral proteins and cell cycle regulation and have been extensively reviewed  and OAS-like protein (OASL) are two related ISGs in humans, which are known to restrict influenza. OAS senses and degrades dsRNA through synthesis of oligoadenylates, which in turn switches on RNase L activity -dependent protein kinase (PKR) is an ISG which functions as both an antiviral effector and anti-proliferative protein during infection  LPAI virus  is highly induced by Type I IFN, and many RNA virus infections. Viperin inhibits IAV by perturbing lipid rafts and thus inhibiting viral budding  are upregulated upon viral infection, and have antiviral activity (Diamond and Farzan, IFITM3 restricts IAV in both duck and chicken cells. Ducks upregulated all IFITMs including IFITM1, IFITM2, and IFITM3 in both lung and ileum during infection with HPAI, whereas chickens showed minimal upregulation of IFITMs (Smith et al., de novo assembly methods on combined RNAseq data. With advances in NGS technology and new software development to analyze fragmented GC rich RNAseq data, we will be able to better mine transcriptomes for genes as well as gain insight into avian immune system evolution. Furthermore, a wealth of genome information from many avian species is becoming available.Throughout this review we have spoken about genes that are presumed missing from ducks, chickens or birds in general. Because bird genomes contain many GC rich areas (Hron et al., In this review, we summarize recent advances in understanding PRR in ducks, comparing them to chicken PRR, and analyzing their downstream signaling adaptors. We also investigated tissue expression of these innate immune components to try to gain insight into where these proteins were most expressed. Higher tissue expression of PRRs and their effectors may allow ducks to respond more quickly to IAV in a tissue-specific manner. A rapid and robust response that is quickly dampened could allow ducks to limit damage from inflammatory sequelae. Duck RIG-I and MDA5 are most highly expressed in the trachea, lung and intestines, areas of both HPAI and LPAI influenza replication . HoweverThroughout this review we summarize the function and regulation of PRRs in chickens, ducks, and humans during IAV infection. While the differences in the RLR pathway are well-studied in ducks, there are currently few studies on TLR and NLR and their adaptor molecules in ducks during IAV infection. As these pathways converge and co-regulate each other, this is a very important piece of the story that is missing. Likewise, many proteins mentioned in this paper have been studied at the regulation level, but very few have been functionally and biochemically characterized.We acknowledge that much of the work is yet to be done characterizing adaptor proteins in IFN and pro-inflammatory cytokine signaling networks. Investigation of these regulatory proteins in ducks and other birds, will allow us to see the conserved mechanisms, and find those that are not. Further, we acknowledge the bias that most immunological research looks at positive regulators of innate signaling. However, as ducks are equally adept at initiating and shutting down inflammatory responses, we should also begin to investigate inhibitory proteins and their expression and function. It should also be noted that functional studies in innate immunity in both ducks and chickens are limited. As such, data on PRR tissue expression and upregulation is often limited to small sample groups. Tissue expression can vary with age and breed of animals, and all studies discussed here used domestic breeds of ducks. When looking at tissue expression of genes as a potential route of resistance, it may be beneficial to also look at gene expression in wild mallards, which are constantly adapting and evolving with IAV. Indeed, it would be worthwhile understanding the allelic diversity of PRR genes and variation in function across many species of wild ducks. This may give us more insight into detection and resistance to IAV in its natural host and reservoir, the mallard duck.LC originally drafted and edited the manuscript. KM edited the manuscript.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.


Notch3 gene knockouts are viable and have limited developmental defects, focussed mostly on defects in the arterial smooth muscle cell lineage. Additional studies have revealed overlapping roles for Notch3 with other Notch proteins, which widen the range of developmental functions. In the adult, Notch3, in collaboration with other Notch proteins, is involved in stem cell regulation in different tissues in stem cell regulation in different tissues, and it also controls the plasticity of the vascular smooth muscle phenotype involved in arterial vessel remodelling. Overexpression, gene amplification and mis-activation of Notch3 are associated with different cancers, in particular triple negative breast cancer and ovarian cancer. Mutations of Notch3 are associated with a dominantly inherited disease CADASIL , and there is further evidence linking Notch3 misregulation to hypertensive disease. Here we discuss the distinctive roles of Notch3 in development, health and disease, different views as to the underlying mechanisms of its activation and misregulation in different contexts and potential for therapeutic intervention.Notch3 is one of four mammalian Notch proteins, which act as signalling receptors to control cell fate in many developmental and adult tissue contexts. Notch signalling continues to be important in the adult organism for tissue maintenance and renewal and mis-regulation of Notch is involved in many diseases. Genetic studies have shown that  Drosophila, which has a single Notch gene [Drosophila Notch. Notch 3 and 4, however, have smaller extracellular domains (ECDs) with 34 and 29 respectively. Further differences are located in the intracellular domain (ICD) which is shorter for Notch3 and 4 and lacks a region thought to act as a transcriptional activator domain (TAD)  . More prDrosophila, depend on endocytosis of full-length Notch and proteolytic activation in the endosomal and lysosomal membranes , that resulted in the expression of the soluble cytoplasmic domain of Notch1, which was constitutively active, linking ectopic Notch activity to cancer progression . While tA number of different factors are reported to drive high Notch3 expression in T-ALL. Activated Notch1 signalling has been shown to drive Notch3 expression, but high Notch3 is also present in T-ALL without activating Notch1 mutations ,57. ReceAberrant Notch3 protein levels in T-All are also associated with altered protein turnover. Histone deactylase 6 (HDAC6) has been shown to have a number of alternative substrates apart from any chromatin modification role . Recent Unlike Notch1, which is mutated to become ligand-independent, Notch3 is rarely mutated in T-ALL . Like otDrosophila Notch, whose endocytic-dependent degradation is dependent on Suppressor of deltex  stress. The latter was mediated by increased expression of ER stress genes, including Bip, and ER-localised chaperones. This increase was abolished by gamma-secretase inhibitors. The intermediate between ER stress and Notch3 appears to be Notch3-driven expression of NADPH oxidase 5 (Nox5), which regulates the production of reactive oxygen species (ROS) [A recently published study further complicates the interpretation of the CADASIL phenotype and provides an alternative explanation . An exames (ROS) . Rho kines (ROS) .A similar conclusion regarding the increased activation of Notch3 in CADASIL was obtained from studies of VSMCs differentiated from induced pluripotent stem cells (iPSCs) generated from the fibroblasts of a CADASIL patient . In thisDrosophila [The nature of the primary defect that leads to Notch3 ECD accumulation is less intensively studied but offers an alternative route to developing a therapeutic strategy if this gap in our knowledge could be filled. Whatever the proposed model, it must take account of the predominance of the cysteine altering mutations associated with the CADASIL disease. The "life cycle of Notch" offers a number of possible points in which Notch3 ECD separation could occur . During osophila ,20. The osophila . Howeverosophila . Anotherosophila . This diosophila . Howeverosophila ,102 and osophila . It was osophila .Given the uncertainties regarding mechanisms of primary defects and how the pathological outcomes arise, some groups have advocated mutation correction as a way forward for therapeutic approaches, including inducing exon skipping to remove the mutant EGF module region . IntriguPulmonary hypertension (PH) results from arterial vessel wall thickening and lumen occlusion in response to increased VSMC and endothelial cell proliferation. It is a progressive disease, which leads to increased pulmonary arterial pressure and death. Notch3 expression is elevated in VSMCs from lung biopsies of PH patients and rat and mouse models with experimentally induced PH . StudiesPH also has a genetic origin and inherited forms are associated with mutations in TGF-β pathway components. However, one study has linked PH to mutations in Notch3 . MutatioNotch3 has perhaps been less intensively studied than other members of the Notch family that have more obviously pleiotropic roles. Nevertheless, the importance of Notch3 to human health is becoming increasingly clear as new developmental functions previously masked by redundancy are revealed and the contribution to adult tissue homeostasis is becoming more appreciated. As with other Notch proteins, Notch3 has many links to human diseases through mutation, altered expression or misregulation of its activity or turnover. The duplication of Notch genes and diversification of their function have likely led to specialisations of particular mechanisms of their regulation. Further studies of the ways in which Notch3 is uniquely regulated compared with other human Notch proteins will underpin the development of means to more specifically target its aberrant activity in different disease contexts.