Fibroblast-like synoviocytes (FLS) play a critical role in the pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment. We analyzed high throughput gene expression and chromatin accessibility data from human and mouse FLS from our and other studies available on public repositories, with the goal of identifying the persistently reprogrammed signaling pathways driven by chronic inflammation. We found that the gene expression changes induced by short-term tumor necrosis factor-alpha (TNF) treatment were largely sustained in the FLS exposed to chronic inflammation. These changes that included both activation and repression of gene expression, were accompanied by the remodeling of chromatin accessibility. The sustained activated genes (SAGs) included established pro-inflammatory signaling components known to act at multiple levels of NF-kappaB, STAT and AP-1 signaling cascades. Interestingly, the sustained repressed genes (SRGs) included critical mediators and targets of the BMP signaling pathway. We thus identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation. We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence. Key features of these cells during homeostasis include the production of extracellular matrix (ECM) components and providing nutrients to the synovial fluid (SF)2. Healthy synovium is composed of multiple layers of FLS, which forms the synovial lining and sublining through cell–cell contacts4. Inflammatory and pro-resolving mediators are tightly regulated to maintain normal synovium functioning1. However, in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), an imbalance between these signals causes homeostasis disruption leading to synovial tissue damage, cartilage destruction and bone degeneration.Fibroblast-like synoviocytes (FLS) are synovial tissue-resident and specialized mesenchymal cells critical for homeostasis4. The transformed FLS are major source of proinflammatory cytokines, cartilage degrading enzymes as well as the osteoblast activating factor and thereby possess an ability to induce synovitis, arthritis and osteoporosis4. Recent genome wide studies have established that multiple signaling and transcriptional factors with potential pathogenic activities exhibit epigenomic reprogramming in the RA FLS7. The inflammatory milieu in RA synovium promotes an abnormal epigenetic landscape, modulates chromatin accessibility and favors the pathogenic processes observed in RA FLS such as invasion, migration and production of proinflammatory mediators9. The vital role of tumor necrosis factor-alpha (TNF) in triggering and maintaining an aggressive FLS phenotype has been well documented both in the in vivo and in vitro studies13. Hence, manipulating TNF-induced sustained chromatin activation in RA FLS represent a treatment opportunity that might aid in combating RA persistence and therapeutic resistance.In the course of RA presentation, persistent inflammatory signals drive the development of an active inflammatory state, which leads to a persistent aggressive phenotype in FLS. These aggressive FLS are often referred to as transformed FLS15. Recent studies suggest that treatment with TNF prolonged chromatin accessibility and histone modifications for up to 72 h in the RA-FLS10. Other studies have also shown that FLS gain a long-term inflammatory memory due to the exposure to TNF15. However, the identity of the genes and the pathways that attain a long-term inflammatory memory remain undefined. Furthermore, the duration for which the inflammatory memory persists is also not well understood. In the present study, using an integrative analysis of our own and publicly available epigenomic and transcriptomic data, we determined the identity of the key signaling pathways that are part of the inflammatory memory and likely contribute to the persistent inflammatory phenotype of RA-FLS. We compared the short-term and chronic TNF-induced transcriptomic and epigenetic reprogramming in mouse and human FLS derived from healthy and chronically inflamed arthritic joints. Our results suggest that chronic exposure to TNF contribute to the development of long-term inflammatory memory and thereby switching FLS from passive responders to imprinted aggressors through the activation and repression of critical signaling networks.Previous studies comparing human RA-FLS to macrophages showed that TNF treatment could induce sustained gene expression in RA-FLS but not in macrophages16. These primary FLS from the Tg197 mice expressed high levels of the human TNF transgene  data from multiple sources; where normal primary mouse FLS (mFLS) between passages 3–5 were treated with or without recombinant human TNF for 16, 24 and 48 h from independent data sets generated in our laboratory and public data sets available on NCBI-Sequence Read Archive  and prostaglandin I2 receptor (PTGIR), pro-angiogenic vascular endothelial growth factor C (VEGFC), a member of the poly(ADP-ribose) polymerase protein family (PARP14) as well as the Fas cell surface death receptor (FAS). We next performed a prediction analysis to determine the transcription factors that are likely to regulate the 52 SAGs, using the transcription factor enrichment analysis ChEA318. This analysis prioritizes transcription factors based on the overlap between given lists of differentially expressed genes based on the putative targets as determined by ChIP-seq experiments from ENCODE projects. The results revealed RELA, STAT1, STAT2, JUN and JUND among the top 10 transcription factors predicted to regulate the expression of SAGs , Birc3 , RELB (encodes for p50 NF-kB transcription factor), FAS (encodes for TNF superfamily cell surface receptor involved in programmed cell death) and IRF1  are examples of components of various proinflammatory signaling cascades. The RNA-seq peak intensities at the exons of each of these genes was higher in both short-term and chronic TNF conditions, compared to the untreated controls. Concomitantly, the intensity of ATAC-seq and DNase-seq peaks at the promoters of these genes was also increased by both short-term and chronic TNF-exposure.In order to determine whether the genes exhibiting sustained differential expression between short-term and chronic TNF-exposure also exhibited sustained changes in chromatin accessibility, we compared the genes assigned to the chromatin accessibility peaks with that of the mSAGs Fig. A. We obsily PARP1 as well ily PARP1 as well BMP4), its downstream transcriptional effector SMAD9  and the BMP signaling target and transcriptional regulator from the family of the inhibitor of DNA binding and cell differentiation proteins (ID4) were a part of this predicted network. CheA3 transcription factor enrichment analysis tool predicted that the transcriptional repressor CCCTC-Binding Factor (CTCF)19 is one of the top candidates likely to downregulate the expression of hSRGs in response to chronic inflammation  mSRGs contained accessible chromatin within 5 kb of their TSS in both short-term and chronic-TNF exposure conditions Fig.  and foun5 mSRGs c20. Epigenetics reprogramming of FLS in particular is suggested to be a major mediator of FLS transformation, RA persistence and therapeutic resistance8. Recent large-scale genome-wide studies have incontrovertibly established that FLS transformation is associated with epigenetic-remodeling. However, which of these changes are reversible and which of them are permanent is not fully understood16. A recent study identified that multiple pro-inflammatory genes in human RA FLS escaped transcriptional repression for at least up to period of 72 h of TNF treatment. These escaped genes were suggested to contribute to the persistence of synovitis observed in RA patients. This study also reported that the reprogramming seems to be restricted to RA FLS and was not observed in macrophages due to their tolerogenic mechanisms, which suppress the expression of TNF-inducible genes14. We aimed to determine whether the absence of tolerogenic mechanisms persists for a long-term and if escape from transcriptional repression can also be observed in FLS from TNF-transgenic mouse model of inflammatory arthritis. The irreversible transformation of FLS behavior directly depends on the signaling cascades and transcriptional networks that are reprogrammed due to the epigenetic mechanisms5. We set out to identify the signaling and transcriptional networks that are specifically susceptible to long-term modulation. It was previously reported that the genomic responses of FLS from TNF-transgenic mice greatly resemble those of RA FLS from humans, suggesting that molecular mechanisms underlying long-term inflammatory memory overlap between mice and humans16. However, FLS from the Tg197 mice differ from human RA FLS, as they continue to express human TNF from the transgene, even under culture condition, where as the human primary RA FLS are not under the direct influence of TNF. We therefore performed a comparative analysis of next-generation sequencing data from 28 different mFLS samples that were subjected to TNF either for short periods or for chronic exposure in the inflamed joints and compared these results with the genome-wide data from 9 human RA FLS samples treated with TNF for varying time periods. Some of these data were generated in our laboratory and others were accessed from the public repositories16. This exercise provided a critical insight into the molecular understanding of the sustained epigenetic remodeling. Importantly, it also led to the identification of the signaling cascades both activated and repressed by these epigenomic changes  and intermediate components such as NF-κB subunit 2 , NF-κB inhibitor alpha  and NF-κB inhibitor epsilon . Other SAGs that directly and indirectly contribute to the sustained and exacerbated NF-κB signaling included the TNFAIP3, which encodes the deubiquitinating enzyme A20, that plays a role in the NF-κB non-canonical activation23. The C–C motif and multiple C-X-C motif chemokine ligands24 and prostaglandin synthesis pathway components were other additional pro-inflammatory pathways that show sustained activation in the RA FLS25. Together these data have uncovered that FLS attain a robust long-term inflammatory memory in response to chronic inflammation. The SAGs identified to possess “long-term inflammatory memory” are likely to govern the aggravated and sustained responses to inflammatory stimuli in the transformed RA-FLS.Our pathway and functional prediction analysis for mSAGs also revealed their strong association with inflammatory diseases and pathways, which aligns with the established roles for pro-inflammatory signaling and transcriptional networks in autoimmune joint diseases. Hence, our data showing that NF-κB, STAT and AP-1 signaling pathways exhibit sustained activation was expected27. A previous study reported that BMP4 is downregulated in the synovial tissue of RA patients28. Others reported that BMP4 plays a critical role in upregulating the expression of SMAD9 due to multiple BMP-responsive elements located at the promoter region of SMAD9 gene29. Further, treatment with exogenous BMP ligands was beneficial as it inhibited the pro-inflammatory phenotype of RA FLS30. Here, we identified that BMP4 and SMAD9 are a part of the long-term inflammatory memory that downregulates their expression. Taken together, we speculate that the sustained repression of BMP signaling may be critically required to ensure the persistently transformed phenotype of the RA FLS.Notably, we observed that chronic TNF induces certain genes to undergo sustained repression, which we refer to as SRGs. At the functional level, the SRGs were predicted to function in pathways regulating stem cell proliferation, growth and differentiation. Interestingly, our study also suggests that SRGs may regulate BMP signaling pathway. In fact, BMP signaling was found to be active in several rheumatic diseases such as oligoarticular juvenile idiopathic arthritis (JIA) and RA. However, the expression of key molecules involved in this signaling pathway seems to differ with disease context and its implications in RA are not completely understoodID4 gene as an SRG. ID4 belongs to the family of the inhibitor of DNA binding domain (ID) containing transcriptional regulators with pivotal roles during development, adult tissue homeostasis and tumorigenesis31. However, their roles in FLS and RA progression are not known. ID proteins are targets of the BMP signaling32, and BMP-SMAD-ID signaling network was previously reported to play a role in tumor growth and angiogenesis35. Interestingly, the BMP-SMAD-ID axis was reported to suppress p16/INK4A-mediated cell senescence during the reprogramming of fibroblasts into pluripotent stem cells36. Since cellular senescence is known to contribute to the increased release of catabolic and inflammation promoting molecules by several cell-types, including the FLS37, our data suggest that the sustained repression of the BMP4-SMAD9-ID4 axis may function as another unique means to bolster the catabolic phenotype of the RA FLS.Another interesting result in our study is the identification of In summary, this study uses bulk RNA-seq data of primary FLS from RA patients and mouse models to show that multiple signaling networks are irreversibly modified due to the TNF-mediated long-term epigenetic and transcriptomic reprogramming. These genetic factors may together contribute to the persistence in FLS transformation and inflammatory joint diseases. Large-scale single cell transcriptomic and epigenetic studies on normal and patient synovial biopsies will further aid in confirming the pathological significance of these findings. The identification of similar mechanisms in mouse and human FLS suggest that mouse models serve as valuable tools to study at least some of the aspects of therapeutic resistance in inflammatory joint diseases. Future studies such as genetic manipulation of the identified SAGs and SRGs in mice will help establish their pathophysiological significance in RA. Therapeutic targeting of the signaling pathways identified in this study could provide a safer alternative to the use of epigenetics modifying drugs, which are often associated with off-target side effects.11. Hind paws from wild-type mice with a C57/B6 background were deskinned, followed by digestion with 1 mg/ml collagenase IV (Sigma-Aldrich) for 2 h at 37 °C under sterile conditions36. Cells were cultured in DMEM with 10% fetal bovine serum (Invitrogen) and utilized at passage-4.All methods were carried out in accordance with the institutional guidelines of Emory University. Animal experiments were approved by the institutional care and use committee (IACUC) at Emory University. FLS were prepared from wild type C57BL/6 mice. FLS were isolated by digesting interphalangeal joint synovium according to previously described protocolp-value < 0.05 (Audic Claverie test) followed by Benjamin-Hochberg multiple testing corrections for determining false discovery rate.Mice fibroblast cells (FLS) treated with 5 ng/mL recombinant human TNF (R&D Systems) for 16 h were harvested at 95% confluence. Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen). Only samples with an RNA integrity number (RIN) > 7.5 were used. Libraries were generated from 250 ng RNA using TruSeq Stranded Total RNA Sample Prep Kit (Illumina). Sequencing was carried out using Illumina Hi-Seq 2500 System . Except for mouse FLS treatment, all other RNA-seq data were downloaded as FastQ files  by PCR amplification using Illumina sequencing adaptor primers (Illumina). Paired-end sequencing was performed on a Hi-Seq 2500 System (University of Georgia Genomics Core) to generate data in FastQ file format. In order to study chromatin accessibility changes due to chronic inflammation, we downloaded FastQ files from a DNase I hypersensitivity sequencing (DNase-seq) experiment of wild-type FLS and inflamed FLS from inflammatory arthritis mouse model  for 16 h and were processed according to standard ATAC-seq protocol described previously39. Functional protein–protein analysis was performed using STRING protein–protein interaction plugin on Cytoscape, an open source software platform for visualizing complex networks pathway analysis tool41. Open-source tool, ChEA3 that uses ChIP-seq experiments from the ENCODE database was used to predict transcription factors associations with SAG and SRGs18.Ingenuity Pathway Analysis tool was used to predict the canonical regulatory pathways and diseases and functions associated with the SAGs and SRGsSupplementary Information.


Drosophila model, we investigated the consequences of a gut chronic dysbiosis on the host physiology. Our results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB activation in the excretory system of the flies is responsible for several of the observed phenotypes. This work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases.Although microbiome–host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the  Among them, Metabolic Syndrome (MetS), a systemic disease also termed as dysmetabolic syndrome, is a world health concern thought to be caused by an underlying disorder of energy utilization and storage that directly increase the risk of diabetes mellitus type 2 as well as cardiovascular and renal diseases4. Although modifications in microbiota have been associated with MetS, it remains unclear whether the presence of keystone bacterial species in the gut is required for triggering the metabolic imbalance associated with the pathologies6. Increasing evidence however suggest that gut bacteria contribute to metabolic disturbances via the stimulation of low-grade inflammation of the gut and distal organs such as lung, brain, liver and kidney8. It is postulated that the translocation of gut bacterial pro-inflammatory metabolites and/or compounds to the circulation could trigger or at least facilitate systemic inflammation and mediate metabolic disorders leading to disease9. In most cases, the nature of the metabolites involved and their mode of action remain unknown.The human intestinal microbiota maintains a symbiotic relationship with its host under normal conditions. However, persistent modifications in the composition and/or in the function of this gut microbial population are associated with several pathologies, including metabolic diseases13. Released by most dividing Gram-positive and Gram-negative gut bacteria, regardless of whether they are symbionts, pathogens, or pathobionts, PGN can be detected throughout the body suggesting a systemic diffusion from the gut microbiota17. In Drosophila, the direct binding of PGN to specific membrane-associated (PGRP-LC) or cytosolic (PGRP-LE) receptors triggers the production of hundreds of NF-κB-dependent immune effectors and regulators that could contribute consequently to inflammatory responses upon gut homeostatic imbalance such as alteration of microbiota20. A growing body of research in both invertebrate and vertebrate models continues to mount that microbiota-derived PGN could have systemic effects on metabolism and behaviors23. However, the causal relationships that underlie the systemic dissemination of PGN in promoting metabolic disorders and subsequent diseases are complex and not yet fully understood.Peptidoglycan (PGN) is a ubiquitous bacteria cell wall component for which the innate immune systems of plants, vertebrates and invertebrates have evolved various classes of sensing pattern recognition receptors among which NODs (Nucleotide Oligomerization Domains) and PGRPs (PeptidoGlycan‐Recognition Proteins) are the most studied27. Therefore, we used a Drosophila model of bacterial enteric dysbiosis to study the consequences of gut-derived PGN on the host’s physiology. A persistent dysbacteriosis can be established in flies by oral infection with the non-pathogenic Gram-negative enterobacteria Erwinia caratovora caratovora (Ecc)29. Sensing of Ecc-derived PGN triggers local and systemic activation of the IMmune Deficiency (IMD) signaling cascade, a NF-κB-dependent pathway that shares similarities with the mammalian Tumor-Necrosis-Factor Receptor1 (TNF-R1) pathway30. Combining phenotypic and comprehensive genetic analysis, we found that chronic systemic inflammation induced by gut-derived PGN causes a wasting syndrome whereby the abdomen becomes swollen due to fluid accumulation and the fat body and ovaries are almost degenerated32. We demonstrate that fluid buildup and fat body wasting are the direct consequence of a NF-κB-dependent local reduction of the insulin/PI3K signaling within the Malpighian tubules, the insect’s equivalent of kidneys. Altogether, this study provides support for the role of PGN as a specific bacteria-derived metabolite contributing to the onset of organ wasting and identifies the Malpighian tubules as major organs integrating immune and metabolic signals.Studies on model organisms have provided valuable insights into genetic diseases in general and in the molecular mechanisms by which the gut microbiota is associated with innate immunity and metabolic disorders in particularEcc14. This entomopathogenic Gram-negative bacteria has been shown to release its cell-wall PGN which represents a strong elicitor of the innate immune signaling cascades in both flies and mammals33. In orally infected flies, gut-born bacteria PGN acts both at a short-range by triggering the IMD/NF-κB pathway in the surrounding enterocytes but also systemically in the whole animal after having crossed the gut epithelium and diffused into the hemolymph15. Consistently, while a short exposure to Ecc led to a rapid and transient activation of the IMD pathway  without consequences on lifespan, persistent recolonization of the gastrointestinal tract with Ecc provoked a constant systemic activation of the NF-κB pathway and a premature death of the flies . Type II cells, also called stellate cells, through which water and chloride ion flow, are interspersed at regular intervals with the type I cells41. Interestingly, activating the NF-κB pathway in adult flies by overexpressing IMD or PGRP-LCa in either cell type using LKRGal4 driver for stellate cells and C42Gal4 driver for principal cells , an organ highly specialized in organic solute transport, metabolism and detoxificationEcc-mediated fluid accumulation and fat body degeneration, we analyzed the immune status of the MTs of Ecc-orally infected flies. Antimicrobial peptide (AMP) targets of the IMD/NF-κB pathway but not of the Toll/NF-κB pathway, were highly induced in MTs 24 h after Ecc oral infection  in MT principal cells could rescue the Ecc-mediated phenotypes. We observed that the Ecc-induced lethality, the bloating phenotype and the fat body wasting but not the degeneration of ovaries were partially rescued by restoring the PI3K cascade in MT principal cells 43 either in the Malpighian principal or stellate cells in non-infected flies reduced life span and provoked a bloating phenotype associated with fat body depletion but did not affect the integrity of ovaries  and TOR (Target of Rapamycin) complex mediate most of the PI3K/insulin signaling metabolic effects3. Recent studies in animal models and in humans support a link between persistent low-grade inflammation caused by microbiota-derived metabolites and various components of MetS-associated chronic diseases48. The identification of these metabolites and their mechanisms of action are therefore critical for uncovering the role exerted by bacterial ecosystems on the setting of metabolic diseases. This study establishes a clear crosstalk between bacteria-derived PGN and an immune NF-κB signaling that contributes to the onset of metabolic disorders in Drosophila. Adult flies chronically infected with the enterobacteria Ecc develop phenotypes that are associated with impaired metabolic homeostasis including fluid buildup as well as fat body and ovary degeneration32. We made the demonstration that systemic activation of the IMD/NF-κB cascade by gut-derived PGN is the main elicitor of these physio-pathologic consequences. Our data clearly indicate that upon chronic enteric dysbiosis, a specific NF-κB-dependent reduction of insulin/PI3K signaling in MTs is the trigger of fluid accumulation and fat body wasting but not ovary atrophy. It is noteworthy that interruption of systemic insulin signaling by ImpL2, which antagonizes insulin signaling by binding to Drosophila insulin-like peptides (DILPs), and bacteria-derived PGN cause a wasting syndrome affecting fluid homeostasis, fat body and ovaries49. Thus, it will be of future interest to determine whether chronic IMD pathway activation in PGN responding organs other than MTs also impact local or systemic insulin/PI3K signaling.MetS, defined by a clustering of metabolic disorders affecting multiple interacting organ systems, is a worldwide health concern with high morbidity and mortality50. Humoral infection with the bacterial pathogen Mycobacterium marinum, a close relative of M. tuberculosis causes a progressive loss of metabolic energy stores in form of fat and glycogen leading to a wasting-like phenotype. This wasting response is in part the consequence of inhibition of the insulin signaling and consequent activation of the transcription factor FoxO51. Finally, activation in the larval fat body of the Toll pathway, the other fly NF-κB signaling cascade, was shown to block insulin-dependent growth and nutrient storage. Since DILP6 is a selective target of Toll signaling, it was proposed that Toll signaling reduces growth by inducing hormone insufficiency in the case of infection52. Whereas most studies have identified the fat body as the main tissue integrating immune and metabolic signals, we showed that the wasting syndrome caused by bacteria-derived PGN is largely fat body independent. Indeed, it is in part the consequence of a PGN dependent insulin signaling reduction in the excretory system highlighting that Malpighian tubules are essential organs for the maintenance of immune and metabolic homeostasis in adult flies. It is interesting to note that PGN from Gram-positive bacteria, called Lys-type PGN is filtered by another component of the excretory system of the insects, the nephrocytes. In flies with no functional nephrocytes, Lys-type PGN accumulate in the hemolymph leading to Toll/NF-kB constitutive activation which is detrimental for the flies53.Previous reports have linked bacteria-immunity and insulin signaling in flies, although most studies have been performed at the larval stage. Persistent activation of IMD was shown to deplete fat reserves, cause hyperglycemia, delay development, and impair larval growth suggesting that IMD influences metabolic regulatory pathways51. In MTs, DILP5 produced by principal cell acts as a local insulin agonist whose expression is modulated upon multiple stress conditions54. Therefore, during enteric dysbiosis, a NF-κB dependent reduction of DILP5 production or release by MTs could locally impair insulin signaling hence affecting renal functions. It could also perturb peripheral target organs for insulin including the fat body. Interestingly, DILP5 production by MTs is under the control of tachykinin, an endocrine peptide produced by the enteroendocrine gut cells in an IMD-dependent manner54.How is IMD activation impairing the insulin pathway in the renal tubules and how the local reduction of this metabolic pathway impacts remote organs such as the fat body remain open questions. One model will involve a modulation of the levels of circulating DILPs. Indeed, although in adult flies the metabolic role of DILPs appears complex, a reduction of expression of these insulin analogs has been suggested to cause infection-induced wasting56. CDK is characterized by a slow and progressive loss of glomerular filtration rate leading to kidney failure and fluid retention57. The complications of this disease also impact beyond the kidney and CKD is associated with multiple physiological and metabolic disturbances including muscle and fat storage wasting58. Based on the relevance of Drosophila MTs as a model for human renal diseases and the conservation of immune–metabolic interactions from invertebrates to vertebrates59, it is tempting to propose that low grade inflammation caused by PGN contributes to the onset of metabolic diseases such as CKD. If so, circulating PGN could be used as an early diagnostic biomarker or a novel therapeutic target to prevent chronic inflammation associated with gut dysbiosis in MetS. The recent validation of the first blocking anti-PGN antibody is, in this respect, an important first step57.Altogether, our results provide compelling evidence that bacteria-derived PGN triggers IMD/NF-κB pathway activation and metabolism disorders in MTs contributing to organ failure and distant fat storage wasting in flies. It is worth to note the remarkable parallels between the results obtained and the physiopathology of the human Chronic Kidney Disease (CKD), a MetS-associated chronic pathology. Mounting evidence show that alterations of gut microbiota play a key role in CKD and cause systemic NF-κB mediated inflammation and alteration of the insulin/PI3K metabolic signalingw1118 (BL #6326) flies was used as the reference control wild-type strain. The PGRP-LBΔ34 and DreddD5560 mutant strains were generous gifts of B. Lemaitre. The following strains were obtained from the Bloomington Stock Center: DaGal4 (BL#55851), R4Gal4 (BL#33832), C42Gal4 (BL#30835), UroGal4 (BL#44416), TubGal80ts (BL#7018 and BL#7019), UAS-PI3KCAAX (BL#8294), UAS-RelishRNAi (BL#28943), UAS-PGRP-LERNAi (BL#60038) and UAS-mCD8RFP (BL#27398). The UAS-FaddRNAi and UAS-PGRP-LCRNAi lines were kindly provided by Meier61, and Lemaitre19. The drivers LKRGal4 and Tret1-1Gal4 were kindly provided by Al-Anzi62 and Schirmeier63, respectively. The UAS-PGRP-LBRD strain was described elsewhere63. The UAS-FoxO™ was kindly provided to our laboratory by Tatar43. The fly stocks used for this study also include MexGal464, UAS-PGRP-LCa37 and UAS-TSC1/2 strains65. Unless otherwise indicated, flies were grown at 25 °C on a yeast/cornmeal medium in 12 h/12 h light/dark cycle-controlled incubators. Flies of genotypes containing the Gal4/UAS/Gal80ts constructs were reared at 20 °C and transferred to 29 °C as 5-day-old adults to allow the expression of the Gal4 transcription factor. The five days of adulthood prior to the onset of Gal4 expression are required to allow maturation of adult structures. Female flies were used for all experiments.The 15.For 1liter of food, 8.2 g of agar , 80 g of cornmeal flour (Westhove Maize H1) and 80 g of yeast extract  were cooked for 10 min in boiling water. 5.2 g of Methylparaben sodium salt  and 4 ml of 99% propionic acid  were added when the food had cooled down. For antibiotic (ATB) treatment, standard medium was supplemented with Ampicillin, Kanamycin, Tetracyclin and Erythromycin at 50 μg/ml final concentrationsErwinia carotovora carotovora 15 (Ecc) cultured in Luria–Bertani (LB) medium at 30 °C overnight. Bacterial cultures were centrifuged at 2,500g for 20 min at RT. Cells were serially diluted in PBS and their concentration was determined by optical density (OD) measurement at 600 nm. For oral infection and subsequent analysis, 5-day-old females raised from egg-to-adult in presence of ATB in the food were used. 24 h before the infection, flies were transferred in vials without ATB and then placed in a fly vial with Ecc contaminated food. This food solution was obtained by mixing a pellet of an overnight culture of bacteria Ecc (OD = 200) with a solution of 5% sucrose (50/50) and added to a filter disk that completely covered the agar surface of the fly vial. To perform survival assays upon chronic infection, flies were infected every 2 days with a fresh solution of Ecc (OD = 200) with a solution of 5% sucrose (50/50). For kinetics involving an acute exposure to Ecc, flies were in contact with infected food for 5 h before being transferred to vials containing a filter disk soaked in a food solution of LB medium with of 5% sucrose (50/50) in presence of ATB. Survival analysis in absence of infection were conducted in vials containing a food solution of LB medium and a solution of 5% sucrose (50/50) in presence of ATB. Dead flies were scored at a set time once a day. A minimum of three vials of 25 flies were used for each survival assay. Experiments were repeated at least twice15. Statistical analyses used one-sided log rank test within Prism (GraphPad software).The bacterial strain used in this study is 2 and observed individually under a stereo microscope. Those displaying a translucent and distended abdomen were scored and transferred to a different fly vial. At least three vials of 25 flies were used for each assay. Statistical analyses used the Fisher’s exact test within Prism (GraphPad software). For neutral lipid staining, carcass/fat body of control and infected flies were dissected in cold PBS, fixed for 30 min in 4% paraformaldehyde on ice, rinse 3 times in PBS and stained with BODIPY 493/503 at 1:1,000 in PBS for 30 min. Ovarian degeneration was determined after dissection by direct observation and quantification of the substantial reduction in ovarian size and loss of mature eggs at 50% of lethality. A minimum of 20 ovaries were analyzed in each assay. Experiments were conducted in triplicate. Statistical analyses used Mann–Whitney test within Prism (GraphPad software). Ovarian health was quantified as the percentage of ovarioles that contained one or more apoptotic egg chamber at stage 9–1049. Briefly, ovaries were dissected in PBS and fixed 30 min in 4% paraformaldehyde on ice. DAPI staining was used as a tool to visualize egg chambers throughout oogenesis and identify those that are dying during mid-oogenesis66. For each experimental condition, a minimum of one hundred ovarioles from at least 20 ovaries were analyzed on fluorescence microscope with UV. Statistical analyses used the Mann–Whitney test within Prism (GraphPad software).The penetrance of the abdominal bloating was analyzed during dedicated kinetics at a set time once a day until all flies were dead. In practice, flies were anesthetized with CO2, transferred to Eppendorf tubes and weighed on a METTLER TOLEDO precision balance.To measure body weight, female flies (n = 24) were anaesthetized with CO15. The amount of mRNA detected was normalized to control rp49 mRNA values. Normalized data was used to quantify the relative levels of a given mRNA according to cycling threshold analysis (ΔCt). Control and experimental conditions were tested in the same ‘run’. Each sample was normalized to its own rp49 control to take into account age-specific changes in gene expression. Results are presented as average and standard deviation of a minimum of three independent experiments. Statistical analyses used Mann–Whitney test, within Prism (GraphPad software).RNA from whole adult animals or dissected organs (n = 15) was extracted with RNeasy Mini Kit . Quantitative real-time PCR, TaqMan, and SYBR Green analysis were performed as previously describedrp49: GACGCTTCAAGGGACAGTATCTG, AAACGCGGTTCTGCATGADiptericin: GCTGCGCAATCGCTTCTACT, TGGTGGAGTGGGCTTCATGMetchnikovin: GATGCAACTTAATCTTGGAGCG, TTAATAAATTTGGACCCGGTCTTGGTTGGCecropin: ATGAACTTCTACAACATCTTCG, GGCAGTTGCGGCGACATTGGCGAttacin A: CCCGGAGTGAAGGATG, GTTGCTGTGCGTCAAGAttacin D: GTCACTAGGGTTCCTCAG, GCCGAAATCGGACTTGDrosomycin: CGTGAGAACCTTTTCCAATATGATG, TTCCACGACCACCAGCATDefensin: GTTCTTCGTTCTCGTGG, CTTTGAACCCCTTGGC4EBP: CATGCAGCAACTGCCAAATC, CCGAGAGAACAAACAAGGTGGPrimers used for qRT-PCR are for:Dissected organs  from 4–6-day old female flies expressing the membrane tagged red fluorescent protein mCD8RFP under the control of the Gal4 drivers used in this work were fixed for 20 min in 4% paraformaldehyde on ice and rinse 3 times in PBT (PBS + 0.3% Triton X-100). DAPI staining was used as a tool to visualize nuclei. Tissues were then mounted in VECTASHIELD (Vector Laboratories) fluorescent mounting medium. Images were captured with a LSM 780 ZEISS confocal microscope and processed using Adobe Photoshop.Supplementary Information 1.Supplementary Information 2.


We then discuss in detail the role of each IFN in controlling pathogen infections in intestinal and respiratory epithelial cells. Finally, we provide our perspective on novel concepts in the field of IFN  that we believe have implications in driving the differences between type I and III IFNs and could explain the preferences for type III IFNs at mucosal surfaces.Interferons (IFNs) constitute the first line of defense against microbial infections particularly against viruses. They provide antiviral properties to cells by inducing the expression of hundreds of genes known as interferon-stimulated genes (ISGs). The two most important IFNs that can be produced by virtually all cells in the body during intrinsic innate immune response belong to two distinct families: the type I and type III IFNs. The type I IFN receptor is ubiquitously expressed whereas the type III IFN receptor’s expression is limited to epithelial cells and a subset of immune cells. While originally considered to be redundant, type III IFNs have now been shown to play a unique role in protecting mucosal surfaces against pathogen challenges. The mucosal specific functions of type III IFN do not solely rely on the restricted epithelial expression of its receptor but also on the distinct means by which type III IFN mediates its anti-pathogen functions compared to the type I IFN. In this review we first provide a general overview on IFNs and present the similarities and differences in the signal transduction pathways leading to the expression of either type I or type III IFNs. By highlighting the current state-of-knowledge of the two archetypical mucosal surfaces ( Interferons (IFNs) were first discovered to interfere with the replication of influenza virus sixty years ago by Isaacs and Lindenmann . Since tThe type II IFN family only has one member: IFN-γ. IFN-γ is produced predominantly by natural killer (NK) cells, natural killer T cells (NKT) and innate lymphoid cells (ILCs) and has been shown to be important for innate and adaptive immune responses . AdditioIn 2003, two groups simultaneously discovered three new cytokines in humans that were able to block viral infection: IL29, IL28A and IL28B also known as IFN-λ1, IFN-λ2 and IFN-λ3, respectively , 17. As e.g. intestine, lung, vaginal, and hepatocytes)  –26 and stocytes) –31.Interferons are produced upon sensing of pathogen associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs). In a simplified view, as this is not the focus of this review, nucleic acids from the viral genome and intermediate products from virus replication are the main PAMPs for viruses. They are recognized by the Toll-like receptors (TLRs) and the RIG-like receptors (RLRs) Figure 1Pathogens can be sensed in different intracellular compartments depending on their mode of entry. In the case of viruses they can either infect the host by directly penetrating the plasma membrane or by being endocytosed and trafficking into the endosome compartment where they will be released into the cytosol . The sital. showed that only a fraction of murine fibroblasts infected with West Nile virus produced IFN mRNA regardless of the viral load  and paracrine manner (the bystander cells) to activate complex signal transduction pathways which will induce transcriptional responses that will ultimately result in the development of an antiviral state in the stimulated cells Figure 2Recent reports have uncovered that while TYK2 is required for type I IFN signaling, it is dispensable for type III IFN signaling , 62. SevFollowing JAK activation and receptor phosphorylation, signal transducer and activator transcription (STAT) proteins are recruited to the complex. STATs are subsequently phosphorylated by JAKs and activated STATs form STAT1/2 heterodimers which bind to IRF9, forming the transcription factor interferon-stimulated gene factor 3 (ISGF3). ISGF3 transfers to the nucleus and binds interferon-stimulated response elements (ISREs), driving the transcription of interferon-stimulated genes (ISGs) Figure 2Probably the most important step in mounting an antiviral response is the ability of cells to turn it off. Failure to arrest IFN signaling in tissues leads to inflammatory disorders in patients and interferonopathies . These din vivo studies using SOCS1 knock-out mice showed increased ISG induction in the liver in response to type III IFNs while the lung and gut were only mildly affected  (e.g. SOCS1 and SOCS3) are considered the most potent negative regulators used by cells to control type I IFN signaling as they can directly interact with TYK2 interfering with its activation . SOCS1 saffected . As lungJAK1 signaling can be regulated by the ISG ubiquitin-specific protease 18 (USP18). USP18 is induced upon both type I and type III IFN treatment, however it specifically regulates type I IFN signaling by binding to IFNAR2 Figure 2Beside activating the JAK/STAT signaling pathway, both type I and type III IFNs also induce the mitogen-activated protein kinases (MAPKs). Interestingly, in human intestinal epithelium cells, both type I and III IFNs activate MAPK signaling pathways however only type III IFNs require them for their antiviral functions. Intestinal cells treated with MAPK inhibitors blocked the ability of type III IFNs to control virus infection while type I IFNs’ antiviral properties stayed intact . These oe.g. murine intestinal cells which lack IFNAR, see section Role of Type I and III IFNs in the Murine Intestine), the properties of the remaining IFN receptor (i.e. IFNLR) could be weakened or enhanced.Interestingly, the signaling pathways downstream type I and III IFNs are interconnected and influence each other . Studiese.g. intestine, lung and liver). These studies have revealed that while there is a core set of ISGs  induced in all tissues evaluated, there are others that may be tissue-specific   , 83, 84.One of the predominant differences between the type I and the type III IFN-mediated immune response is that both cytokines induce ISG expression with very different kinetics. Human intestinal epithelial cells treated with either IFN-β1 or IFN-λ1-3 were shown to induce a similar set of ISGs but these ISGs were induced with different magnitudes and at different times post-IFN stimulation . Type I i.e. Mmp7, Serpinb1a, and Csprs)  . These Id Csprs) .Our current understanding of type I and III IFN-mediated signaling suggest that while the main signal transduction pathways are very similar between both IFNs, there are unique differences between each cytokine Table 1 vice versa)  . Howevere versa) . Importae versa) . A simile versa) , 90. In e versa) , 90. Mate versa) . It is iThis heterogeneity in ISG induction is not exclusively found in type I IFNs, as recent studies have shown that 90% of a clonal population of mouse derived IECs responded to IFN-β1, whereas 55% of these cells responded to type III IFNs . This diFurthermore, as type III IFNs act on epithelial surfaces it is important to consider their polarization state. Many experiments in laboratory settings use epithelial cells in sparse conditions whereas in the normal tissue environment they are tightly connected and polarized. The state of the cells is critical when evaluating responsiveness to IFNs as mouse intestinal cells have shown to be more sensitive to IFN-λ when reaching a polarized status  while huThe main difference that places type I and type III IFN apart lies in the fact that the type III IFN receptor expression is restricted to a subset of cells , providide novo virus production and damage to the intestinal epithelium  and that depletion of IFN-λ signaling from IECs resulted in an increase in norovirus, rotavirus and reovirus replication and fecal shedding   have passed clinical trials and have been approved for use in treating virus infections , multiple sclerosis, leukemia, melanoma, and multiple myeloma . Current. USP18) . Current. USP18) , 116.in vitro they regain responsiveness to type I IFNs : project numbers 415089553 (Heisenberg program), 240245660 (SFB1129), 278001972 (TRR186), and 272983813 (TRR179) to SB; 416072091 to MS. We also acknowledge funding from the German Academic Exchange Service (DAAD) (Research Grant 57440921) to PD and the China Scholarship Council to CG.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.


RAF1 and BRAF) as well as multiple receptor tyrosine kinases [Sorafenib is an oral multikinase inhibitor approved for the treatment of unresectable hepatocellular carcinoma, differentiated thyroid cancer, and advanced renal-cell carcinoma. This drug blocks tumor cell proliferation and angiogenesis by inhibiting the Raf serine/threonine kinases RAF and BRAF kinases . Of thesA 62-year-old man was referred to us for evaluation of 2 exudative lesions on his lower extremities of 1-month duration. His medical history included sorafenib 400 mg/day for 4 months for diffuse hepatocellular carcinoma. The patient denied a history of similar lesions prior to the onset of sorafenib. Physical examination revealed 2 indurated erythematous plaques on both thighs that were 2 to 6 cm in diameter . The lesSkin swabs for viral and bacterial cultures were negative. A skin biopsy showed perifollicular and perivascular lymphohistiocytic infiltration . DilatedFive cases of acne inversa-like lesions associated with sorafenib have been reported, all of them in males ,2. CliniIf we apply the Naranjo drug reaction assessment tool to this case, the result would be “probable.” Unlike previously reported cases, our patient developed acneiform lesions while on a low dose of 200 mg twice a day. Further studies are needed to elucidate the pathogenic explanation and dose relationship between sorafenib and acne inversa-like lesions.


Tropheus and Aulonocara. Within each pair, taxa differed in terms of red and yellow body colouration, and high‐performance liquid chromatography (HPLC) analyses of skin extracts revealed different carotenoid profiles and concentrations across the studied taxa. Five genes were differentially expressed in all three yellow–red skin contrasts , but only the tetratricopeptide repeat protein-coding gene ttc39b, whose gene product is linked to mammalian lipid metabolism, was consistently expressed more highly in the red skin samples. The RNA-Seq results were confirmed by quantitative PCR. We propose ttc39b as a compelling candidate gene for variation in animal carotenoid colouration. Since differential expression of ttc39b was correlated with the presence/absence of yellow carotenoids in a previous study, we suggest that ttc39b is more likely associated with the concentration of total carotenoids than with the metabolic formation of red carotenoids.Carotenoid pigments play a major role in animal body colouration, generating strong interest in the genes involved in the metabolic processes that lead from their dietary uptake to their storage in the integument. Here, we used RNA sequencing (RNA-Seq) to test for differentially expressed genes in a taxonomically replicated design using three pairs of related cichlid fish taxa from the genera  Animals acquire carotenoids from their diet, modify them enzymatically and deposit taxon- and tissue-specific mixtures of carotenoids in integumentary tissues such as skin and feathers . The resCarotenoid pigments are also involved in the outstanding body colour diversity among cichlid fish in African and South American waters ,10. In fttc39b, in the red skin samples in each contrast. A previous study associated ttc39b with avian bill colour polymorphism [ttc39b in carotenoid colouration across vertebrate classes.In the present study, we used RNA sequencing (RNA-Seq) to screen for genes associated with carotenoid colour differentiation among three pairs of related cichlid taxa, which differ in terms of red and yellow carotenoid-based body colouration . This demorphism , and our2.n = 6 males per colour variant) as indicated in Tropheus moorii ‘Mbita' versus T. moorii ‘Moliro', (ii) T. sp. ‘black' ‘Ikola' versus T. sp. ‘black' ‘Bulu Point', all four from Lake Tanganyika and (iii) Aulonocara baenschi versus A. hansbaenschi ‘Red Flush’ from Lake Malawi (Tropheus population pairs (T. moorii and T. sp. ‘black') diverged 1–2 Mya, and divergence between Tropheus and Aulonocara is 5 Mya [We sampled yellow and red skin patches from three pairs of related cichlid taxa .First-strand cDNA for quantitative PCR (qPCR) was synthesized from 500 ng RNA of each skin sample extract using the high capacity cDNA reverse transcription kit (Applied Biosystems) and diluted 1 + 3 for the subsequent qPCR reactions. Primer design and the qPCR protocol are described in the electronic supplementary material. Relative expression levels (RQ) were determined by the 2−ΔΔCq method  using gegeiger [Associations between gene expression levels and skin colour  were tested by phylogenetically controlled ANOVAs using the R-package geiger . The phyn = 3 males per colour variant), with skin samples taken from the same body region on the other side of the fish. Samples were extracted twice in a solution of acetone with butylated hydroxytoluene  and analysed on an Agilent 1290 UHPLC System with an Agilent Zorbax Eclipse Plus C18 . Signals were recorded at 480 nm. Integrated peak areas from the two consecutive extractions were summed for the assessment of signal strength, relative to skin sample fresh weight, as a proxy of the relative carotenoid content in the skin sample.Integumentary carotenoids were extracted from some of the same fish as used for RNA-Seq . Among the differentially expressed genes in each taxon pair, only five genes showed significant expression differences between yellow and red skin samples in all three colour contrasts: dhrsx, nlrc3, tcaf2, urah and ttc39b . Of these, only ttc39b expression was consistently regulated in each taxon pair, showing significantly higher expression levels in the red skin tissues relative to the yellow tissues (a). Two genes, dhrsx and tcaf2, showed congruent expression patterns with TmY < TmR, TbY > TbR and AbY < AhR. Differential expression of urah followed the pattern TmY < TmR, TbY > TbR and AbY > AhR, and that of nlrc3 was TmY < TmR, TbY > TbR and AbY > AhR (a).High–performance liquid chromatography (HPLC) analysis of skin extracts confirmed the presence of free and esterified carotenoids in both yellow and red coloured cichlid variants. Carotenoid profiles varied between colour variants, and integrated peak areas were larger in the red skin samples compared to the contrasted yellow variants . In each cichlid pair, expression levels of ttc39b were significantly higher in the red compared to the yellow skin (b). Similarly, qPCR confirmed the RNA-Seq results for the other three genes, except for a non-significant result for dhrsx in the comparison between TbY and TbR .The RNA-Seq results were validated by qPCR analysis of low skin b. Similanlrc3, tcaf2 and urah. By contrast, expression levels of dhrsx  and ttc39b  showed significant covariation with colour across taxa .In accordance with the prevalence of taxon pair-specific expression patterns, phylogenetically controlled ANOVAs across all taxa revealed no significant associations between skin sample colour and expression levels of 4.ttc39b in the skin of three pairs of closely related cichlid fish taxa, which differed in red versus yellow body colouration and skin carotenoid content. Since our experimental fish had been fed identical diets, differences in integumentary carotenoid content must have been produced by metabolic processes. Currently, there is no functional evidence for a connection between ttc39b expression and carotenoid colouration, but the role of its gene product in cholesterol and lipid metabolism [ttc39b interferes with Liver X Receptor (LXR) signalling [npc1l1 expression [ttc39b with changes in human blood lipoprotein levels [ttc39b on carotenoid uptake and storage.We report consistent differential expression of tabolism  suggeststabolism ,13. The tabolism ,22. The gnalling  and may pression ,24. The n levels  and withttc39b has been detected in previous RNA-Seq studies concerned with body colour variation. These studies reported the elevated expression of ttc39b in the orange compared to the white skin regions of one of two examined clownfish species [ttc39b from previous studies, and combining it with the strong signal of ttc39b in our dataset, we propose ttc39b as a compelling, novel candidate gene for carotenoid colouration.Differential expression of  species , in the  species  and in y species . Further species . Collatettc39b with yellow–red colour variation that we observed here and that has previously been described in the estrildid finch [ttc39b in the metabolic formation of red carotenoids. This interpretation is also compatible with increased expression of ttc39b in the reddish-orange clownfish skin [ttc39b in the yellow compared to non-carotenoid coloured skin detected in other fish studies [ttc39b was not related to variation in integumentary triglyceride content. We therefore consider it possible that ttc39b expression covaries with integumentary carotenoid concentration rather than composition in this and the previous studies. Quantitative comparisons of total carotenoid concentrations are confounded when samples contain different types of carotenoids, as is the case in our study . Furthermore, although all skin samples were treated equally, extraction efficiencies may nevertheless have varied among samples. Nonetheless, a crude approximation by HPLC peak area integration indeed points towards higher carotenoid concentrations in the red than in the yellow skin samples across the investigated cichlid variants, congruent with the variation in ttc39b expression levels .The association of id finch  hints toish skin . It is,  studies ,26, in p studies  were domttc39b—also demonstrated taxon pair-specific patterns of differential gene expression. Together, these data hint at variation in the molecular mechanisms behind the expression of carotenoid colour diversity in cichlid fish.The differences among the HPLC chromatograms of the skin carotenoid extracts imply different biochemical backgrounds of the yellow–red colour contrasts represented by the three taxon pairs. This conforms with the gene expression data, which—except for ttc39b in the carotenoid-based colouration of both birds and fishes suggests a conserved function of the gene in vertebrate colouration.Another tetratricopeptide repeat protein, RCP2, was recently found to regulate carotenoid accumulation in the floral tissues of monkeyflowers . While T


Cruciform DNA is a causing factor of genome instability and chromosomal translocation, however, most studies about cruciform DNA in mammalian cells were based on palindromic sequences containing plasmids and reports about endogenous cruciform DNA are rare. In this study we observed the dynamics of endogenous cruciform DNA in mouse growing oocytes using immunofluorescence labeling method. We found cruciform DNA foci exist in transcription active growing oocytes but not in transcription inactive fully grown oocytes and colocalized with Parp1 but not with DNA damage marker γH2A.X. By analyzing the Genotype-Tissue Expression data, we found cruciform DNA-mediated chromosomal translocation in human spermatocytes is associated with the specific DNA transcription in testis. When inhibiting the transcription with α-amanitin in mouse oocytes, we found oocyte cruciform DNA foci decreased significantly. In summary, we observed the endogenous cruciform DNA in growing oocytes and our results showed that the cruciform DNA formation is transcription-dependent. When double-strand DNA which contain directed repeats are denatured, the intra-strand indirect repeats in both strands can anneal and form stem or stem-loop secondary structures. Then the stems or stem-loops with the up- and down-stream double-strand DNA will form a new specific four-way junction structure in DNA called cruciform DNA . The forAlthough cruciform DNA is a common structure in genomic DNA and causing factor for human diseases, which factors promote cruciform DNA formation and how cruciform DNA induce genome stability are still not well understood. On the one hand, the formation of cruciform DNA induced DNA double-strand breaks (DSBs) might be cell-specific and be an instantaneous event, so we still don’t know at which time and in which cell the cruciform DNA is formed and in what conditions the cruciform DNAs could be transformed into DSBs . On the Unlike somatic cell and spermatocyte, mammalian growing stage oocyte has relatively larger nucleus, which is termed as germinal vesicle  and convAll animal manipulations in this study were performed according to the animal experiment standards and guidelines of Guangdong Second Provincial General Hospital and approved by the Ethical Committee of Guangdong Second Provincial General Hospital. All oocytes manipulations in this study were under the oocyte manipulation media M2  with 2.5 μM milrinone  to block the oocytes from meiosis resumption. To isolate the large antral follicle oocytes, ovaries of 18 days post partum (dpp) or 8-weeks old ICR strain female mice were firstly collected. Then ovaries were chopped with blade and oocytes were isolated with mouth pipette. To isolate the pre-antral follicle oocytes, we collected pre-antral follicles from chopped mouse ovaries and treated the follicles with Trypsin-EDTA solution  at 37°C for 25 minutes and removed the somatic cells by mouth pipette.To inhibit the RNA polymerase II mediated transcription in oocytes, oocytes were treated with 25 μg/mL α-amanitin  for 6 hours. If the oocytes were used for labeling the newly synthesized RNAs, the oocytes were treated with 0.5 mM 5-ethynyl uridine (EU)  for a further 2 hours.The antibodies used in this study are: cruciform DNA antibody [2D3] ; γH2A.X antibody ; and Parp1 antibody . Oocytes were firstly fixed with 4% Paraformaldehyde Fix Solution  at room temperature (RT) for 20 minutes and permeated with 0.3% Triton at RT for 20 minutes. Then oocytes were treated with Quick Antigen Retrieval Solution for Frozen Sections  at RT for 45 minutes. After washing 2 times, oocytes were blocked with 1% BSA for 1 hour and incubated with primary antibodies at 4°C overnight. After washing 3 times, oocytes were incubated with secondary antibodies and then stained with Hoechst and mounted on glass slides. Then oocytes were observed under the Dragonfly Confocal system.If EU click reaction was needed, we shorted the fixation and permeation time to 15 minutes and the antigen retrieval time to 10 minutes. After washing of secondary antibodies, we preformed the EU click reaction according the manual of BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 647  but changed the reagent EdU with EU.The transcriptome data used in this study are originated from the Genotype-Tissue Expression (GTEx) project  and downStudents’ t test was used in this study to assess the significance of the foci number differences in oocytes. Fisher’s exact test was used to assess the significance of the oocyte proportion of different groups. P-value < 0.01 was marked by **.To analyze the localization and dynamics of cruciform DNA in mouse growing oocyte, we collected different stage oocytes from preantral follicles and large antral follicles of 8 weeks old mice. By immunofluorescence labeling of the cruciform DNA with antibodies we found that cruciform DNA could be detected in preantral follicle oocytes, NSN and NSN-SN (the transition stage of oocytes from growing NSN state to fully grown SN state) stage oocytes but not in the fully grown SN oocytes . The aveEvidences had suggested that Parp1 could bind at the ends of hairpin arms of cruciform DNA on plasmids . In thisAs DNA transcription is the main cause factor of negative supercoiling in DNA , we nextIn addition to the analysis of GTEx data, we also analyzed the effects of DNA transcription on the cruciform DNA formation in oocytes. We chose 18 dpp old mouse oocytes for this analysis as most of these oocytes are either NSN oocytes or NSN-SN oocytes which are transcription active, cruciform DNA foci positive and easier for us to do statistical analysis. When we treated the 18 dpp mouse oocytes with RNA polymerase II inhibitor α-amanitin –31, we fIn this study, we analyzed the dynamics of endogenous cruciform DNA in growing oocytes. As we known, a single cruciform DNA molecule is hard to observe under confocal microscope, so we think the bright cruciform DNA foci detected by antibodies might be tandem cruciform DNA structures but not be only one single cruciform DNA structure. Indeed, in mammalian genome, there are lots of microsatellites which are AT-rich  and thesAs cruciform DNA will increase the risk of genomic instability, so cells need to resolve the hairpin structures on cruciform DNA to decrease the risk of DNA damage. However, how the hairpin structures on cruciform DNA are resolved by cells were still not well known. Using palindromic sequences containing plasmids, structure specific DNA endonucleases had been found being essential for cells to resolve the cruciform DNAs . In our From our results we also found the formation of cruciform DNA is strongly associated with DNA transcription. Our results indicated that spermatocyte specific transcription of adjacent regions of palindromic sequences (such as PATRR11 and PATRR22) might be the causing factor of the cruciform DNA-mediated genomic instability. This result also suggested that the t formed in human sperm is transcription-dependent but not meiosis-dependent.As a conclusion, we observed the endogenous cruciform DNA foci in growing oocytes and the formation of cruciform DNA is transcription-dependent. However, which factors cause the genome instability at cruciform DNA region is still need to be investigated. Lastly, the growing mouse oocytes could be used as a model to analyze the cruciform DNA mediated genome instability and Parp1 could be used to verify or live-cell observe the cruciform DNA in growing oocytes.


Patients undergoing haemodialysis (HD) are at higher risk of developing worse outcomes if they contract COVID-19. In our renal service we reduced HD frequency from thrice to twice-weekly in selected patients with the primary aim of reducing COVID 19 exposure and transmission between HD patients.Dialysis unit nephrologists identified 166 suitable patients (38.4% of our HD population) to temporarily convert to twice-weekly haemodialysis immediately prior to the peak of the COVID-19 pandemic in our area. Changes in pre-dialysis weight, systolic blood pressure (SBP) and biochemistry were recorded weekly throughout the 4-week project. Hyperkalaemic patients (serum potassium > 6.0 mmol/L) were treated with a potassium binder, sodium bicarbonate and received responsive dietary advice.There were 12 deaths (5 due to COVID-19) in the HD population, 6 of which were in the twice weekly HD group; no deaths were definitively associated with change of dialysis protocol. A further 19 patients were either hospitalised and/or developed COVID-19 and thus transferred back to thrice weekly dialysis as per protocol. 113 (68.1%) were still receiving twice-weekly HD by the end of the 4-week project. Indications for transfer back to thrice weekly were; fluid overload (19), persistent hyperkalaemia (4), patient request (4) and compliance (1). There were statistically significant increases in SBP and pre-dialysis potassium during the project.Short term conversion of a large but selected HD population to twice-weekly dialysis sessions was possible and safe. This approach could help mitigate COVID-19 transmission amongst dialysis patients in centres with similar organisational pressures. Severe Acute Respiratory Syndrome Coronavirus-2 has developed into a worldwide pandemic, with over 44 million documented cases and 1.1 million deaths worldwide. Medical comorbidities such as hypertension, diabetes mellitus, asthma, obesity and chronic kidney disease are reported as significant predictors of morbidity and mortality in COVID-19 patients [The necessary frequency of haemodialysis (HD) is particularly pertinent at a time of a worldwide pandemic . There iDialysis reduction in selected patients was performed in our centre prior to the peak incidence of the COVID-19 pandemic in the North West of England, UK. The primary aim was to reduce COVID 19 exposure, transmission between patients and allow for social distancing whilst on the HD unit. It also permitted the formation of a ‘hot’ dialysis unit to cohort all suspected, proven cases and contacts for COVID-19, whereas non-exposed patients could be grouped into ‘cold’ satellite dialysis units. At the onset of this project we began collecting data in a structured way and this paper describes our methodology, outcomes and learning.> 6.5 mmol/L was the only absolute indication to convert back to thrice weekly dialysis. Increased surveillance of dialysis parameters were indicated if any of the following events occurred: SBP > 180 mmHg, UFR > 10 mL/kg/hr., pre-dialysis weight > 5% of target weight and > 3 l ultrafiltration (UF) per session. Dietetic consultation and medical review of both dialysis sessions in that week were undertaken. Pre-dialysis potassium was monitored closely with the following management plan adopted:Our regional renal service has a catchment population of 1.55 million people and undertakes in centre haemodialysis for 432 patients in one main centre and 4 outlying satellite centres. The furthest distance between the main centre and satellite centres is 18 miles, which facilitated re-designation of patients without excessive travel times or major inconvenience. In centre HD patients were remotely reviewed  by their own nephrologists to determine suitability for twice weekly dialysis  were not changed during this period. All patients were prescribed 4 h of dialysis.Demographic, comorbidity, biochemical and dialysis adequacy comparisons between patients who continued to receive thrice weekly dialysis and those transferred to twice weekly dialysis were performed. Diagnosis, medication and comorbidity data were taken from coded diagnoses available on hospital and primary care electronic medical records. Usual pre-dialysis blood pressures were calculated using the average of the last readings prior to 3/23/20. Urine output was not contemporaneously measured but patients who self-reported anuria (< 100 mL per day) was recorded. Thirteen patients who commenced twice weekly dialysis after being identified later on during the course of this project were subsequently excluded from analysis in the thrice weekly group at baseline.Patients who were hospitalised or admitted to the COVID-19 dialysis ‘hot unit’ were transferred to thrice weekly dialysis for the rest of the project. All hospitalisations were recorded after scrutinizing hub hospital (where the main centre is based) admission data and all referrals from surrounding district hospitals for the period 3/23/20 to 4/20/20. Patients were defined as COVID-19 positive after a positive nasal and/or throat COVID-19 PCR. Patients who were transferred back to thrice weekly dialysis completed 3 dialysis sessions in the following 7 days. Cause of death was obtained from death certification and all fatal cases were discussed at a weekly multi-disciplinary mortality and morbidity meeting (including at least 5 nephrologists) to evaluate whether the reduction in dialysis frequency was contributory.This is an observational report of a strategic and organisational restructuring of dialysis provision in our centre during the COVID 19 pandemic..Data were analysed using means and standard deviations (parametric data) and medians and interquartile ranges (non-parametric data) where appropriate. Categorical data were compared using chi square test. Continuous data were compared using unpaired T test and Mann U Whitney. Significance of pre-dialysis SBP, potassium and weight changes were compared at each data point  using Mann-Whitney U test. Data were analysed on an as-treated basis. Only patients participating in twice weekly dialysis at each time point were analysed.From a cohort of 432 in-centre dialysis patients 179 (41.4%) were identified as potentially suitable for and agreed to undergo twice weekly dialysis. This manuscript reports on the 166 (38.4%) patients who then commenced twice weekly dialysis on 3/23/20. Patients who were transferred to twice weekly dialysis were more likely to be older, with lower ultrafiltration volumes, greater urea reduction ratio (URR), shorter dialysis vintage and to have lower pre-dialysis phosphate and potassium levels. There was no significant difference in the frequency of primary renal diseases between the two groups and there were significantly less patients with heart failure transferred to twice weekly HD , hyperkalaemia despite use of binders (n = 4), patient’s request (n = 4) and compliance concerns (n = 1). The characteristics of these 28 patients who we know “failed” twice weekly dialysis due to definite dialysis related reasons (rather than exposure or contraction of COVID-19 for example) are displayed in Table We determined this by including patients who died (6), patients automatically transferred back to thrice weekly dialysis because of hospitalisation 13) or suspected/confirmed COVID-19 infection (6) as “failing twice weekly dialysis”. There were 113 (68.1%) patients who were able to continue twice weekly dialysis for the whole 4 week period . 7/19 patients were subsequently transferred back to thrice weekly dialysis and 1 patient did not tolerate either medication. Nine patients continued to take the medications and dialyse twice weekly. Hyperkalaemia interventions reduced pre-dialysis potassium by 0.9 mmol/L (IQR 0.8–1.4); one patient’s potassium increased following intervention.This analysis found that the majority of patients who are deemed suitable to temporarily convert to twice weekly dialysis were able to safely dialyse twice weekly for at least 1 month during the COVID-19 pandemic, enabling safer grouping of patients to reduce potential viral exposure and transmission and ease service demands which may have been exacerbated by staff sickness. However, this was only possible with very close monitoring via dedicated clinician time and through the use of digital technology allowing remote monitoring of biochemistry and dialysis parameters. The necessity for close monitoring can be demonstrated for two reasons. Firstly, longitudinal assessment of dialysis parameters demonstrated statistically significant increases in pre-dialysis systolic blood pressures and pre-dialysis potassium in those patients who continued to receive twice weekly dialysis, although overall these parameters remained well within ‘safe’ limits. Secondly, it was also noted that the rate of patient transfer back to thrice weekly dialysis was constant throughout the project at around 3–4% per week.Rising SBP despite no significant increase in pre-dialysis weight suggests that blood pressure changes were not necessarily related to increases in extracellular blood volume (ECV). This finding is not surprising given that more frequent dialysis has been shown to improve blood pressure control through various mechanisms. These include reduced ECV, increased sodium removal, reduced sympathetic tone and removal of vasoactive factors which may be driving hypertension –17. TherThere was little difference in the UF volumes in the twice weekly patients at baseline compared with those still maintaining on the twice weekly protocol at the end of the 4-week period (1.4 (1.0–2.0) litres per session compared with 1.5 (1.0–2.0) litres per session). However, this excludes the 19 13.5%) patients who were transferred back to thrice weekly because of fluid-related issues. Table .5% patie< 5.4 mmol/L at the end of this project. This was the same as in the thrice weekly population. Only 2 of the 6 patients with this degree of hyperkalaemia had previously had a pre-dialysis potassium above 6.0 mmol/L during the entire project. We had made no changes to dialysate potassium concentrations because recent evidence has suggested a higher mortality risk when patients are dialysed against a low potassium dialysate (1mEql/l), particularly those patients with a higher serum potassium [Due to reduced weekly dialysis time it is unsurprising that the median pre-dialysis potassium significantly increased every week. A pre dialysis potassium > 6.0 mmol/L has been suggested as a threshold whereby mortality risk substantially increases . Howeverotassium . The useotassium . Potassiotassium .There were no significant differences between hospitalisations, COVID-19 infections and deaths between the two groups although the twice weekly group were on average 6 years older than the thrice weekly group, perhaps representing a more at-risk group, although they had lower prevalence of heart failure. There was no definite evidence that any of the 6 deaths in the twice weekly dialysis group were caused by a reduction in dialysis frequency. There were no hospitalisations for fluid overload in the twice weekly group.The analysis of this 4-week period of change in dialysis protocol is not intended to re-energise the debate over long-term dialysis frequency and dialysis dose but provides a potential methodology to appropriately and safely rationalise dialysis resources during a health service crisis such as the current pandemic. Although we are not recommending generalisation of our approach as health care management differs markedly throughout the world, it may have implications for other countries where dialysis resources are limited. Although we did not have definitive inclusion criteria, we have retrospectively surveyed the clinicians involved and together with the findings of this study we would suggest that short term switch to twice weekly dialysis is most appropriate for;We investigated complete conversion of patients to twice weekly dialysis. Future studies could investigate the impact of alternating twice to thrice weekly dialysis in selected groups of patients to minimise COVID-19 exposure but maintain satisfactory dialysis parameters. At this stage, having passed the first peak of the COVID-19 pandemic in our geographical region, all our in-centre haemodialysis patients are now being individually reassessed for their suitability and preference for either home haemodialysis or peritoneal dialysis, the latter with particular relevance to patients with residual urine output currently treated with twice weekly haemodialysis. Patients who will not be suitable or do not wish to be transferred to home therapies and do not have significant formally quantified residual renal function will return to thrice weekly dialysis in a planned fashion over the next few weeks .In conclusion, the COVID-19 pandemic is an international exceptional health crisis. This project demonstrates that reorganisation of dialysis provision for selected patients has the potential to protect patients and clinical staff. Patient selection and careful real-time monitoring can ensure that abrupt changes in dialysis provision are safe.This analysis did not fully evaluate dialysis adequacy which is reported to influence long term outcomes of HD as this project was primarily designed to overcome the short-term challenge of dialysis provision during a pandemic. It was not possible to perform contemporaneous measurements of residual renal function due to the short lead time of project set-up and there was considerable missing data regarding anuric status (47.6%). Regular measurement of residual renal function within the whole dialysis cohort would undoubtedly aid in decision making to determine suitability to convert to twice weekly haemodialysis at short notice. There was no measure of concordance with interventions (i.e. compliance with potassium binder medication) for hyperkalaemia although all but one patient’s potassium fell after its initiation. Whilst patient’s views were continually monitored during this period there were no quality of life or intradialytic health related quality of life symptoms recorded throughout the 4-week period.


Nasopharyngeal carcinoma (NPC) is an important cancer in Hong Kong. We aim to utilise liquid biopsies for serial monitoring of disseminated NPC in patients to compare with PET-CT imaging in detection of minimal residual disease.Prospective serial monitoring of liquid biopsies was performed for 21 metastatic patients. Circulating tumour cell (CTC) enrichment and characterisation was performed using a sized-based microfluidics CTC chip, enumerating by immunofluorescence staining, and using target-capture sequencing to determine blood mutation load. PET-CT scans were used to monitor NPC patients throughout their treatment according to EORTC guidelines.The longitudinal molecular analysis of CTCs by enumeration or NGS mutational profiling findings provide supplementary information to the plasma EBV assay for disease progression for good responders. Strikingly, post-treatment CTC findings detected positive findings in 75% (6/8) of metastatic NPC patients showing complete response by imaging, thereby demonstrating more sensitive CTC detection of minimal residual disease. Positive baseline, post-treatment CTC, and longitudinal change of CTCs significantly associated with poorer progression-free survival by the Kaplan–Meier analysis.We show the potential usefulness of application of serial analysis in metastatic NPC of liquid biopsy CTCs, as a novel more sensitive biomarker for minimal residual disease, when compared with imaging. This greatly reduces survival and the quality of life. NPC occurs with a peak age for diagnosis only in the upper 40s, when an individual is still in the prime of life. In Hong Kong young males less than 44 years of age, NPC is the top cancer in terms of incidence.2 Earlier diagnosis and better predictive markers for metastatic tumours are important in the quest to improve patient survival.3 However, the survival for NPC patients with recurrent or primary metastatic disease is dismal, with a median overall survival of 20 months.4Nasopharyngeal carcinoma (NPC) has its highest incidence in Southern China, including endemic regions like Hong Kong.5 Detection of rare CTCs in the blood provides an exciting opportunity for early sensitive detection of metastatic cancer cells. The clinical utility of CTCs to monitor treatment response and prognostication has been demonstrated for various metastatic cancers.12 The challenging task of enrichment and isolation of rare CTCs in the blood has greatly improved and heralds an exciting opportunity to utilise blood-based cancer diagnostics to monitor cancer progression and treatment response real-time.14 The technological advancement for use of microfluidics approaches and hydrodynamic centrifugal forces allows fast and efficient size-based isolation of viable and label-free CTCs after depletion of white blood cells.15 We embarked on pilot studies for CTC detection and enumeration for six cancer types including breast, lung, colorectal, gastric, liver and prostate cancer patient bloods. Our previous work established succinct workflows for CTC enumeration after enrichment.16 This CTC isolation system and platform provides a “liquid biopsy” for monitoring disease progression and offers the opportunity of non-invasive real-time monitoring of patients to determine the efficacy of treatment regimens.Most epithelial cancer patients die from distant metastasis. Circulating tumour cells (CTCs) may be considered as seeds of metastasis due to escape of the cancer cells into the bloodstream from both primary and metastatic tumours.19 However, CTC studies of NPC are scanty.11 To date, there are no studies for serial longitudinal examination of patients during treatment to reveal the dynamics and characterisation of the molecular features of CTCs for NPC patients undergoing palliative chemotherapy. We assessed the usefulness of CTC enumeration before, during and post-treatment to complement the currently utilised plasma EBV and PET-CT imaging for assessment of treatment outcome. We determined whether the mutation load obtained by NGS analysis after CTC enrichment is useful as a biomarker for detection of minimal residual disease. This current study provides the first NPC serial CTC analysis study utilising the size separation and CD45 depletion approach for 21 metastatic NPC patients. We now show the potential clinical utility of CTC analysis and the more sensitive detection of minimal residual disease in patients with partial or complete responses at the end of treatment in this serial study of metastatic NPC patients accompanied by PET-CT imaging for assessment of tumour burden and plasma EBV DNA levels. Liquid biopsies are the source of CTCs and plasma EBV DNA, which provide complementary clinical utility for early detection of minimal residual disease in metastatic NPC patients to enable clinicians to implement more radical treatment regimens to improve patient treatment outcomes.NPC is a radiosensitive tumour. However, for patients with late diagnosis and lymph node metastasis, survival prospects remain poor. Distant metastasis increases mortality. Monitoring multiple lesions and distant metastatic solid tumours in advanced metastatic NPC patients by conventional invasive biopsies is not practically feasible and is inherently limited due to the intrinsic tumour molecular heterogeneity. Thus, we aimed to perform molecular analysis of CTCs to assess the clinical utility of non-invasive real-time monitoring of disease treatment in metastatic NPC. Numerous clinical studies utilise the plasma EBV DNA test for prognostication of advanced NPC.From 2015 to 2018, 21 NPC patients with distant metastasis planned for palliative chemotherapy were prospectively recruited from three hospitals, Queen Mary (QM), Queen Elizabeth (QE) and Pamela Youde Nethersole Eastern (PYNE) Hospitals. Serial blood samples were taken at four different timepoints before, during and at the end of treatment, as shown in Fig. 20 The output CTC enriched sample was loaded and fixed on a poly-l-lysine slide for subsequent enumeration of CTCs by immunofluorescence (IF) staining according to standard protocols. CTC isolation and enumeration were performed with pan-keratin (CK), which recognises keratins 4, 5, 6, 8, 10, 13 and 18. Cells were pre-hybridised with blocking solution and then hybridised to a cocktail of antibodies of pan-CK/EpCAM  and CD45  conjugated with Alexa-555 and APC secondary antibodies and stained with DAPI, as previously described. The cell images obtained from metastatic NPC sample analysis were captured on a fluorescence scanner, the Cytation 5 Cell Imaging Multi-Mode Reader . The imaged CTCs were counted by imaging software using CellProfiler and CellProfiler Analyst, as previously described.16 CTCs were defined as nucleated cells positive for DAPI and CK/EpCAM IF staining, but negative for CD45 IF staining.Collected blood samples were directly put into STRECK tubes and processed within 72 h. CTC enrichment utilised CTChip®FR1 biochip analysis performed on a ClearCell®FX1 System  after lysis of red blood cells in 7.5 ml blood samples and centrifugation at 500 g at room temperature for 10 minutes, followed by resuspension of cell pellets.21 The real-time PCR assay targeting the Bam HI-W region of the EBV genome was performed in duplicate and calibrated with an internal DNA standard using Namalwa, which is an EBV-positive cell line harbouring two copies of the EBV genome per cell.23 In blood sampling timepoint EBV1, a level of 1500 copies/ml was considered significantly elevated from the baseline blood of NPC patients for CTC analysis, as previously reported in an earlier NPC CTC study.11 In blood sampling timepoints EBV2–4, a cut-off of 60 copies/ml was used to define positive EBV status, as was consistently detected by all laboratories joining the NRG-HN001 NPC study from the National Cancer Institute.24For CTC samples collected for the serial analysis, the plasma EBV DNA was quantified by real-time PCR with the LightCycler 480 system (Roche), as previously described.25 KYSE270 cells and a Red Cross healthy donor’s blood DNA sample (RC2202) were used in the spike-in experiment. To explore the sensitivity of our NGS protocol for a scenario with low purity of CTC content, mixtures of different proportions of KYSE270 DNA to RC2202 DNA ranging from 100%, 10%, 5%, 2.5%, 1.25% and 0% were subjected to whole-genome amplification (WGA), according to the Qiagen REPLI-g Single Cell WGA protocol. An indexed library was constructed and hybridisation to the Roche Nimblegen NGS SeqCap EZ capture kit for target capture analysis and pair end 150 lllumina Hi-seq sequencing was performed, as previously described.26 This custom kit covers up to 7 Mb of 1366 genes, which are commonly involved in oncology, particularly in metastasis, and as druggable targets. The sequencing data were processed following the GATK recommendations.27 In brief, the sequencing reads were aligned to human genome (hg19) by Burrows-Wheeler Aligner (BWA) followed by the INDEL realignment and base quality recalibration. The quality of the sequencing data is summarised in Table 26 The germline variants including SNVs and small INDELs were detected using GATK HaplotypeCaller. The workflow for NGS mutational profiling was as previously published.26 For clinical CTC samples, 7.5 ml blood was utilised for CTC enrichment by the high purity run protocol according to the manufacturer instructions. The output was subjected to the NGS assay as described above for WGA, library preparation and bioinformatics analysis. The combined annotation dependent depletion (CADD) score was used to assess damaging effects of missense mutations.28 Only the damaging protein-altering mutations, missense mutations defined by the CADD score >15 were included in blood mutation load. Validation of mutations detected in CTC samples was performed by Sanger sequencing for 87.8% (35/41) of the tested mutations.DNA extracted from an oesophageal squamous cell carcinoma (ESCC) cell line (KYSE270) was utilised in this spike-in experiment. ESCC cell lines were cultured in RPMI, as previously described.p < 0.05 was considered statistically significant.The receiver operating characteristic (ROC) analysis was run to test the sensitivity and specificity of CTC1-4, EBV1-4 for progression-free survival (PFS) of mNPC. The CTC numbers and log10 transformed EBV DNA copy number/ml were used as continuous variables for the ROC analysis. Spearman’s rank-order correlation was used to assess the relationship between different CTC counts and log10 transformed EBV DNA amounts at different timepoints. Overall survival (OS) and PFS were measured from the date of baseline blood collection before palliative CT treatment to the last disease evaluation or until death/recurrence. The interim imaging assessment was scheduled midway during the treatment. Kaplan–Meier curves of probability of PFS were used to analyse survival data and examined the association of clinical parameters and biomarkers with PFS and OS. During the evaluation of the markers of change of EBV DNA, CTCs or combined both CTC4/EBV4, patients with positive status at both biomarkers, either one marker, or negative status at both, were classified as group 2, 1 and 0, respectively. Statistical analyses were performed by SPSS version 26 and a 16 Figure Previously, we established the CTC enrichment protocol based on cell-size separation, which is able to isolate viable and label-free CTCs in our laboratory using a spiral microfluidics CTC chip and the downstream workflow for CTC enumeration.The cell line spike-in quality control validation experiment demonstrated our established NGS protocol and pipeline was reliable for detecting the SNVs with a good sensitivity, positive predictive value (PPV > 97.0%), and low false discovery rate (FDR < 3.0%) from 10% down to 5% purity. The known mutation landscape of KYSE270 detected before and after WGA, and the mutations detected in various concentrations of KYSE270 in the background healthy donor RC2202 are shown in Supplementary Fig. 29 The results of serial analysis of EBV plasma, CTC counts, and imaging for all patients are summarised in Table 11 was positive for 17/21 (81%) cases before chemotherapy (CT) (EBV1). The EBV plasma assay using a lower stringent cut-off of 60 copies/ml for timepoints EBV2-4 was positive for 13/20 (65%) cases after two cycles of CT (EBV2), for 11/19 (57.9%) cases after three cycles of CT during treatment (EBV3) and for 11/20 (55%) patients at the end of treatment (EBV4). CTC enumeration, using the cut-off of ≥1 CTCs as positive, showed positive results for 11/20 (55%) patients at CTC1 before CT, 10/20 (50%) patients after two cycles of CT (CTC2), 11/18 (61%) patients at CTC3 after three cycles of CT, and 14/21 (67%) patients at the end of treatment (CTC4). The NGS blood mutation load analysis used a cut-off of >0.57 mutations per Mb based on our cell line spike-in experiments to detect positive minimal residual disease. We identified 13 NPC patients with positive blood mutation loads at the end of treatment by the NGS blood mutation load analysis.Among 21 metastatic patients, 11 (52.4%) were newly diagnosed patients suffering from synchronous distant metastasis at the time of diagnosis (i.e. IVC disease), while 10 patients developed de novo distant metastasis after initial radical treatment. The most common metastatic sites were bone, lung, liver and lymph nodes (LNs) Table . After aLiquid biopsy longitudinal real-time disease monitoring with the EBV assay, the CTC enumeration or NGS analysis was able to track the tumour burden in the nine patients (1–9) with poor treatment outcome Table . PatientPET-CT imaging indicated four NPC patients had a partial response at the end of treatment; patient 11 was concordantly positive for tumour burden with both EBV and CTC findings, while patients 10, 12 and 13, in contrast, were three PMR patients with a negative EBV status during and at the end of treatment Fig. . Two indImaging assessment at the end of CT detected complete response for eight NPC patients 14–21), but of these, four patients relapsed after 8–24 months (patients 14–17)  with post-treatment radiologic imaging showed CMR and negative evidence of NPC disease from plasma EBV DNA, but the tumour burden was supported by the positive CTC analysis, remained progression-free for 1.5 years. The post-treatment blood mutation load of patient 19 was 22.9 mutations/Mb. The post-treatment CTC count of patient 21 was positive. These two patients require a longer follow-up to verify the post-treatment positive CTC mutation load suggestive of minimal residual disease.Two NPC patients (18 and 20) had a CMR with the longest disease-free survival (28 and 30 months) and negative testing results from both plasma EBV DNA and CTC liquid biopsies for disease burden .The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CTCs and EBV DNA load in mNPC with PFS are summarised in Fig. p = 0.014, EBV3, p = 1.3 × 10−5, and EBV4, p = 2.8 × 10−5) , whereas CTC2, CTC3, and blood mutation load at CTC4 did not reach statistical significance  , CTC2/CTC3 (ρ = 0.663), CTC3/CTC4 (ρ = 0.692), EBV1/EBV2 (ρ = 0.653), EBV2/EBV3 (ρ = 0.850), EBV3/EBV4 (ρ = 0.862), EBV1/EBV4 (ρ = 0.661). The correlation between CTC1/CTC4 was low (ρ = 0.117). The high-risk group includes patients with detectable CTCs at both timepoints, the low-risk group includes patients with undetectable CTCs at both timepoints, and others were classified as intermediate-risk group. The same procedures were performed with EBV DNA changes and results are shown in Fig. A Spearman’s rank-order correlation was run to assess the relationship between baseline, during and post-treatment CTC counts in twenty-one mNPC cases. There were statistically significant positive correlations between CTC1/CTC2 ρ = 0.56, CTC2/CT14 We recently established a platform for isolation of label-free CTCs based on size separation. We now demonstrate the feasibility of utilising liquid biopsies for CTC enumeration and NGS analysis to obtain the blood mutation load and comparative plasma EBV assay for monitoring the efficacy of chemotherapeutic treatment in NPC patients having distant metastasis. The EBV plasma status collected at the middle and end of treatment was a good independent predictive biomarker for treatment response representative of the tumour bulk and significantly associated with PFS in metastatic NPC patients.30 We validated previous observations that plasma EBV DNA findings provide good prognostic information of PFS and treatment outcomes of distant metastasis in NPC. The liquid biopsy analysis by CTC enumeration or blood mutation loads detected minimal residual disease in 75% (6/8) NPC patients  at the end of palliative CT, showing CMR by imaging. Post-treatment plasma EBV findings detected minimal residual disease in 62.5% (5/8) NPC patients with marginally positive readings of 7, 119, 53, 35 and 4 copies per ml . Our studies show that liquid biopsy CTC molecular findings provide a more sensitive means for earlier detection of residual tumour burdens compared to imaging. The current study for the first time suggests the potential clinical application of examining the blood mutation load of CTCs and serial CTC enumeration, which can complement commonly utilised plasma EBV DNA detection used at the completion of chemotherapy for patients with metastatic NPC. This is particularly relevant for the management of those patients found to have negative or only low-positive EBV DNA levels just marginally raised above the cut-off level. Our findings show the post-treatment CTC test is a potentially more sensitive tool to detect minimal residual disease in those good responders with a negative EBV test  within 6 months compared to those with complete response . Future studies with larger patient cohorts including advanced locally recurrent NPC patients at stages III, IVA and IVB are ongoing and will be useful to determine the clinical utility of blood-based real-time monitoring of tumour progression for personalised medical care of advanced NPC patients.Recent CTC studies now show that CTC enumeration in breast, colon, and prostate cancer patients are sensitive indicators of disease status and are even more sensitive and quantitative than conventional MRI and PET-CT scans or serological detection of cancer biomarkers.36 Although nine  metastatic NPC patients had zero CTC counts before treatment, this finding may be attributed to the lack of detection by immunocytochemistry of CTCs not expressing epithelial markers for CTCs likely undergoing EMT35 or for CTCs of smaller size, which may not be captured. This also partially explains the discrepancy between the post-treatment CTC counts and blood mutation loads for some patients. One possible reason for finding two positive post-treatment CTC enumerations with negative blood mutation load may be due to fragility of CTCs, which may compromise DNA integrity and hinder their capture for NGS analysis. Further efforts for the search of tumour-specific markers are necessary for improvement of the CTC enumeration sensitivity and specificity.Mechanistic CTC studies from glioblastoma, breast and liver cancers, suggested aggressive subpopulations of CTCs detected in the blood circulation have invasive mesenchymal characteristics.The current data suggest that CTC analysis can provide complementary information for detection of minimal residual disease, when the EBV results are ambiguous and PET imaging indicate PMR or CMR at the end of the palliative CT for NPC patients with disseminated disease. Based on the current dataset, we find that CTC analysis at the end of treatment is useful for sensitive minimal residual disease detection and warrants further validation in a larger sample cohort with longer follow-up times to determine its clinical usefulness. The current study is limited by the small sample size and the follow-up time. We report data as a pilot study for generation of the hypothesis for further validation with larger sample size on the potential clinical utility of CTC analysis for good responders of metastatic NPC. The current study establishes a genomic profiling approach for CTCs by NGS target capture sequencing for identification of the molecular signature for metastatic progression and tracking molecular events occurring during the tumour evolution under treatment pressure. Genomic mutational analysis of CTCs will provide strategic information on key genetic alterations hallmarking metastatic tumours. Serial monitoring of patients during treatment will provide useful information on tumour evolution and the development of drug resistance. Incorporation of the non-invasive liquid biopsy CTC assay may obviate the need of invasive tissue biopsy and radiological imaging for monitoring and predicting cancer progression and for guiding treatment choices. Our long-term goal is to improve real-time treatment monitoring and risk stratification strategy to offer clinicians vital timely information to tailor treatment regimens for individual NPC patients. We expect the NGS mutational profiling capability to enhance our future understanding of the genetic basis for metastatic disease and identify predictive molecular signatures associated with drug resistance and poor treatment outcomes.Supplementary document