Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease with potentially severe complications including cirrhosis and hepatocellular carcinoma. Previously, we have identified circulating lipid signatures associating with liver fat content and non-alcoholic steatohepatitis (NASH). Here, we develop a metabolomic map across the NAFLD spectrum, defining interconnected metabolic signatures of steatosis .We performed mass spectrometry analysis of molecular lipids and polar metabolites in serum samples from the European NAFLD Registry patients (n = 627), representing the full spectrum of NAFLD. Using various univariate, multivariate, and machine learning statistical approaches, we interrogated metabolites across 3 clinical perspectives: steatosis, NASH, and fibrosis.Following generation of the NAFLD metabolic network, we identify 15 metabolites unique to steatosis, 18 to NASH, and 15 to fibrosis, with 27 common to all. We identified that progression from F2 to F3 fibrosis coincides with a key pathophysiological transition point in disease natural history, with n = 73 metabolites altered.Analysis of circulating metabolites provides important insights into the metabolic changes during NAFLD progression, revealing metabolic signatures across the NAFLD spectrum and features that are specific to NAFL, NASH, and fibrosis. The F2–F3 transition marks a critical metabolic transition point in NAFLD pathogenesis, with the data pointing to the pathophysiological importance of metabolic stress and specifically oxidative stress.NCT04442334).The study is registered at Clinicaltrials.gov  in the blood with the aim of providing a comprehensive molecular overview of fat build-up, liver fibrosis, and diagnosed severity. We identify a key metabolic ‘watershed’ in the progression of liver damage, separating severe disease from mild, and show that specific lipid and metabolite profiles can help distinguish and/or define these cases.  •We assembled a highly phenotyped and characterised cohort of patients with NAFLD across the full spectrum of NAFLD.•Lipidomic and metabolomic interrogation of this dataset reveals crucial metabolic tipping point of NAFLD at fibrosis stage F2–F3.•NAFLD processes of fibrosis, steatosis, and NASH progression parallel distinct changes in metabolomic profiles.•en route to late-stage NASH.Oxidative stress-buffering potential of the liver via ether lipids appears 1 of the key changes  Non-alcoholic fatty liver disease (NAFLD) is a chronic disease, characterised by hepatic triglyceride accumulation exceeding ∼5%, progressing in some to non-alcoholic steatohepatitis (NASH) and advanced fibrosis/cirrhosis.NAFLD is associated with several comorbidities including components of metabolic syndrome , cardiovascular disease, end-stage liver disease, and hepatocellular carcinoma (HCC). Given the intricate links between NAFLD and wider metabolic diseases, NAFLD should be considered part of a broad multisystem disease state.e.g. amino acids and intermediates in energy metabolism) are those involved in primary, central metabolism and thus of high relevance to the liver metabolism and NAFLD.Histological assessment remains the reference standard for assessing NAFLD.The scientific understanding of NAFLD has improved in recent years regarding pathophysiology, genetics, transcriptomics, and metabolomics (including lipidomics).NCT04442334). This cohort includes the full spectrum of disease, from histologically normal liver tissue through NAFL to NASH-F4 (cirrhosis). The protocol and detailed description of the Registry has recently been published.Samples came from a cohort of 627 histologically characterised patients recruited from clinics at a number of leading international tertiary liver centres  participating in the ‘European NAFLD Registry’ project  were formalin-fixed and paraffin-embedded. Tissue sections (3–4 μm thick) were stained with H&E and trichrome stain for visualising collagen. All cases were recruited at tertiary centres where liver biopsies were routinely assessed according to accepted criteria by experienced liver pathologists and scored using the well-validated National Institute of Digestive Diseases and Kidney NASH Clinical Research Network (NIDDK NASH-CRN) system.Analyses of molecular lipids and polar metabolites are described in detail in the 2-transformed, and scaled to zero mean and unit variance. The sample intersection between the lipidomic, polar metabolomic, and clinical information datasets was retained. A detailed description of the statistical analysis methodology in provided in the All analyses were carried out in the R statistical programming language environment .Following integration of the metabolomics and clinical data, the study comprised 627 histologically characterised individuals across the full spectrum of NAFLD severity , with a r = -0.13), NAFLD grade  and fibrosis stage (r = -0.19). Likewise, the ether PCs (PC(O)s; LC6) connected to 6 clinical variables, including to fibrosis (r = -0.12) and NASH (r = -0.12). Additionally, 2 further metabolites were linked directly with fibrosis: 3-OH-butanoic acid (r = 0.20) and glycerol-3-phosphate (r = -0.10). Whereas 3-OH-butanoic acid also linked with diagnosis of T2DM (r = 0.21), glycerol-3-phosphate did not attain such a link in this analysis. Conversely, glycerol-3-phosphate weakly associated with ballooning of cells in the liver (r = -0.03), whereas 3-OH butanoic acid did not. These interactions are also depicted in the correlation plot , in which clear groups of lipid classes are visible . The polp <0.05) in either an ANOVA or Tukey honest significant difference (HSD) analysis. It should be noted that, as log2-transformed and autoscaled data are used throughout all heat maps and intercomparisons for calculation of changes, the calculated changes function as fold change on a log2 scale, as opposed to any direct changes in units of concentration. For the grade of disease activity using the binary categories of NAFL vs. NASH, as there are only 2 disease states, results are represented as a volcano plot  is summarised in ano plot C. The fuObserving the cumulative number of changing lipids and polar metabolites across fibrosis progression, using F0 as a reference point, a total of 9 metabolites changed by F1, 33 by F2, 77 by F3, and 83 by F4 A, therebp <0.05) differing levels, by ANOVA or Tukey HSD, between participants across steatosis, NAFL/NASH grade, and fibrosis stage were tabulated  across (1) fibrosis stages and (2) steatosis grades, and (3) between NAFL vs. NASH are given in For visualisation at an individual level, a selection of representative lipids and polar metabolites found to change significantly . There was little overlap between steatosis and NAFL/NASH, while excluding fibrosis, adding only 4 TGs to the signature, for a total of 6 TGs.Differentiating only between NAFL and NASH, and excluding changes across steatosis and fibrosis, a signature of (n = 18) lipids was found, including 9 TGs (increased in NASH), 2 sphingomyelins (SMs), 6 PC(O)s, and 1 phosphatidylethanolamine .In total, 8 lipids and 7 polar metabolites were found to change solely with fibrosis stage, namely PC(32:1), PC(35:4), PC(37:4), PC(40:5), PC(40:6), SM(d36:1), SM(d36:2), and SM(d38:2), and the polar metabolites were 2-hydroxybutanoic acid (2-HB), 3-hydroxybutanoic acid (3-HB), citric acid, isoleucine, lysine, and oleic acid.Consistent with the frequent co-existence of steatohepatitic and fibrotic processes as disease evolves, a considerable overlap between associated metabolites was observed, with 46 lipids and 1 other metabolite  identified. The lipids were 29 TGs, 8 PCs, 3 LPCs, 3 PEs, 2 SMs, and 1 DG.At the core of all 3 histological disease components, 27 lipids, but no polar metabolites, were found to change with increasing disease severity. This group of lipids is dominated by 20 TGs, followed by 5 LPCs and 2 PEs.In total, 3 Venn diagrams  show theTo further characterise the metabolic evolution of NAFLD, 2 well-described disease outcome deflection points were studied, comparing mild disease with either ‘clinically significant’ fibrosis (NASH F2–F4) or ‘advanced’ fibrosis (NASH F3–F4),The goal of this study was not to develop biomarkers but to examine pathophysiological changes as the disease evolves. However, as an exemplar of how the metabolomic signatures may be leveraged, median AUCs during recursive feature addition to generate minimal feature sets for both discrimination tasks are shown for the lipid and metabolite (LM) analyses in vs. F2–F4 task and F0–F2 vs. F3–F4 tasks, respectively. These compared favourably with models built using standard clinical data points used in clinical practice , which achieved median AUCs of 0.746 (95% CI 0.056) and 0.778 (95% CI 0.051). Combining clinical features to the lipids and polar metabolites (LMC) produced minor improvement in median AUC (+0.004 and +0.022), showing competitive performance of lipids and polar metabolites with the top clinical features. The relative performances of the models built with all input predictor sets  for both classification tasks  using our minimal feature sets are given and contrasted in Discriminative models using lipids and polar metabolites only (LM) achieved median AUCs of 0.732  and 0.765  for the F0–F1 Our results provide a detailed overview of lipids, polar metabolites, and key clinical variables and observations in NAFLD, providing potential insights into the metabolic basis of pathophysiology and disease natural history. As a key novel finding, our analysis pinpoints a critical transition point in the metabolic profiles of patients with NAFLD, concurrent with the progression from fibrosis stage F2–F3. Interestingly, this increase in fibrosis stage is also the stage at which both all-cause and liver-related mortality begin to markedly increase,We identify a set of circulating TGs, PCs, Cers, and 1 CE that appear to pertain solely to hepatic steatosis burden, and not to NASH or fibrosis progression. Given the hepatotoxicity of Cers and the increasing understanding of their role in cellular damage in NAFLD,de novo lipogenesis,Despite the expected overlap in metabolic signature between NAFL to NASH transition and fibrosis progression, there remained 18 lipids associated solely with the presence of NASH, independent of fibrosis stage. We identify a signature of increases in 9 TGs and decreases in 6 PCs, 2 SMs, and 1 PE, which are specific to NAFL–NASH conversion. It must be noted that the presence of an NAFL to NASH conversion-unique signature, apparently distinct from the signature of the progression of fibrosis may be caused by potentially increased statistical power of the ANOVA/Tukey HSD tests on lipids and metabolites between NAFL and NASH, given that these groups contained larger numbers of samples, not being spread across the 5 stages of fibrosis (F0–F4). Regardless, this metabolic signature may be capturing clinically informative metabolic changes that warn of NAFL to NASH progression but that occur independent of steatosis or fibrosis. The TGs that increase in NASH include those TGs with low carbon number and double bond count, which were previously found to be associated with As compared with other assessments performed at biopsy , fibrosis stage is considered the most tractable surrogate marker of likely clinical outcome in NAFLD.In this work, we identified a clear metabolic tipping point at the F2–F3 transition in this cohort of patients with NAFLD. We also demonstrate a fibrosis-specific metabolic signature and a steatohepatitis signature, albeit with some overlap between these.,vs. female assignment of all lipids/metabolites, would give 44 (45%) male-unique, 20 (21%) shared, and 33 (34%) female-unique in fibrosis, 15 (25%) male-unique, 18 (31%) shared, and 26 (44%) female-unique in steatosis, with 82 (68%) male-unique, 16 (22%) shared, and 12 (10%) female-unique in NAFL/NASH. Despite these clear sex differences, the ‘both sexes’ analyses identified 75 of 97 (77%) of those lipids/metabolites agreed upon by the separate male and female analyses in the case of fibrosis, 35 of 59 (59%) for steatosis and 92 of 120 (77%) for NAFL/NASH. The full list of lipid/metabolite direction changes and significances for fibrosis, steatosis, and NAFL/NASH are given for all 3  analyses in vs. F3–F4 , we observed increases in several PCs and TGs and decreases in PC(O)s, LPCs, and SMs, as well as several metabolites including 2- and 3-HB. Increased 2-HB was previously found as an early indicator of insulin resistance,We acknowledge that the detailed identification of the underlying mechanisms driving the metabolic transition observed at F2–F3 awaits further study. Knowledge of these metabolites’ changes and the underlying metabolic pathways may also help in further refining the staging of NAFLD as well as provide targets for future pharmacological interventions. Furthermore, although our study suggests that lipids/metabolites may be useful in the discrimination of patients before, or across, this metabolic ‘tipping point’ (F2–F3), establishing biomarker performance was outside the scope of this study, and further prospective studies will be needed to assess and validate the potential diagnostic value of the metabolites identified. This is currently being assessed within the EU Innovative Medicines Initiative (IMI2) Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) project.Taken together, our findings, from an initial top-down approach, provide a comprehensive overview of the metabolic network across the full spectrum of NAFLD, integrated with clinical measures and assessments. We go on to provide high-resolution metabolomic interrogation of NAFLD, both as a whole and then, uniquely, from 3 different crucial perspectives of the disease, namely, steatosis, NAFL–NASH conversion, and fibrosis, identifying metabolic signatures unique and common between each, even in light of the sexually dimorphic nature of the disease. We additionally observe a potentially important metabolic tipping point at the F2–F3 transition, where our data support the role of metabolic stress and specifically oxidative stress. Notably, F2–F3 progression associates with several metabolic markers previously associated with development of type 2 diabetes. Finally, we demonstrate utility of our lipidomic and polar metabolomic datasets in comparison with established clinical measures in discriminating between patients with NAFLD before or after this metabolic tipping point, suggesting a potential use in clinical practice.Elucidating Pathways of Steatohepatitis (EPoS) consortium funded by the 10.13039/100010661Horizon 2020 Framework Program of the European Union under Grant Agreement 634413; the Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) consortium funded by the 10.13039/501100010767Innovative Medicines Initiative (IMI2) Program of the European Union under Grant Agreement 777377; this Joint Undertaking received support from the 10.13039/100010661European Union’s Horizon 2020 Research and Innovation Programme and 10.13039/100013322EFPIA; and the 10.13039/501100012295Newcastle NIHR Biomedical Research Centre and the European NAFLD Registry.This study received support from the Proposed and designed the study: MO, QMA. Supervised the study: MO, QMA, AKD, TH. Performed data analysis: AM. Performed metabolomics analysis: DG. Supervised metabolomics analysis: TH. Contributed to clinical data acquisition in the European NAFLD Registry cohort: VR, MA, JB, SP, CO, EB, JMS, QMA. Coordinated clinical data acquisition in the European NAFLD Registry cohort: QMA. Wrote the manuscript: AM, MO. Provided further major contributions to the manuscript: QMA, AKD, OG, TH. Critically revised the manuscript for intellectual content and approved the final manuscript: All authors.The guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis: MO.https://www.metabolomicsworkbench.org/) at DOI https://doi.org/10.21228/M85976.Data from this study are available from the Metabolomics Workbench (QMA is Coordinator of the EU IMI-2 LITMUS consortium, which is funded by the EU Horizon 2020 programme and EFPIA. He reports research grant funding: Allergan/Tobira, AstraZeneca, GlaxoSmithKline, Glympse Bio, Novartis Pharma AG, and Pfizer Ltd. Consultancy: 89Bio, Allergan/Tobira, Altimmune, AstraZeneca, Axcella, Blade, BMS, BNN Cardio, Cirius, CymaBay, EcoR1, E3Bio, Eli Lilly & Company Ltd., Galmed, Genentech, Genfit SA, Gilead, Grunthal, HistoIndex, Indalo, Intercept Pharma Europe Ltd., Inventiva, IQVIA, Janssen, Madrigal, MedImmune, Medpace, Metacrine, NGMBio, North Sea Therapeutics, Novartis, Novo Nordisk A/S, PathAI, Pfizer Ltd., Poxel, ProSciento, Raptor Pharma, Roche, Servier, Terns, The Medicines Company, and Viking Therapeutics. Speaker: Abbott Laboratories, Allergan/Tobira, BMS, Clinical Care Options, Falk, Fishawack, Genfit SA, Gilead, Integritas Communications, Kenes, and Medscape. Royalties: Elsevier Ltd.Please refer to the accompanying ICMJE disclosure forms for further details.


Little information is available concerning protein expression of the calcitonin receptor-like receptor (CALCRL) at the protein level. Here, we developed a rabbit monoclonal antibody, 8H9L8, which is directed against human CALCRL but cross-reacts with the rat and mouse forms of the receptor. We confirmed antibody specificity via Western blot analyses and immunocytochemistry using the CALCRL-expressing neuroendocrine tumour cell line BON-1 and a CALCRL-specific small interfering RNA (siRNA). We then used the antibody for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic tissues. In nearly all tissue specimens examined, CALCRL expression was detected in the capillary endothelium, smooth muscles of the arterioles and arteries, and immune cells. Analyses of normal human, rat, and mouse tissues revealed that CALCRL was primarily present in distinct cell populations in the cerebral cortex; pituitary; dorsal root ganglia; epithelia, muscles, and glands of the larger bronchi; intestinal mucosa (particularly in enteroendocrine cells); intestinal ganglia; exocrine and endocrine pancreas; arteries, capillaries, and glomerular capillary loops in the kidneys; the adrenals; Leydig cells in the testicles; and syncytiotrophoblasts in the placenta. In the neoplastic tissues, CALCRL was predominantly expressed in thyroid carcinomas, parathyroid adenomas, small-cell lung cancers, large-cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear-cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours with strong expression of CALCRL, the receptor may represent a useful target structure for future therapies. CALCRL enrichment was conducted using wheat germ lectin agarose beads , as previously described . SubsequFor adsorption controls, the anti-CALCRL antibody was preincubated for 2 h at room temperature with either 10 µg/mL of the immunising peptide (peptide 2) or 10 µg/mL of a control peptide that corresponded to a different region of the receptor .Endogenous CALCRL-expressing BON-1 cells (DSMZ) were seeded onto coverslips and grown to 80% confluence. The cells were then either left untreated or treated with chemically synthesised, double-stranded CALCRL siRNA duplexes (Santa Cruz Biotechnology) in accordance with the manufacturer’s instructions. A scrambled siRNA was used as the negative control (Santa Cruz Biotechnology). Next, the cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 20 min at room temperature. After the fixed cells had been thoroughly washed with phosphate-buffered saline, they were incubated with the anti-CALCRL antibody 8H9L8 (1:500 dilution) overnight at 4 °C, then incubated with the Alexa Fluor 488-conjugated anti-rabbit secondary antibody  for 2 h at room temperature. Finally, the fixed cells were mounted using Fluoromount G (Invitrogen) and analysed using an LSM 510 META laser scanning confocal microscope . To establish controls for immunostaining analyses, the anti-CALCRL antibody was either omitted or adsorbed for 2 h at room temperature with 10 µg/mL of the immunising peptide (peptide 2) or with 10 µg/mL of a control peptide that corresponded to a different region of the receptor .Double-labelling immunocytochemistry with CALCRL and RAMP1, RAMP2, or RAMP3 was conducted by first incubating fixed cells with the anti-CALCRL antibody 8H9L8 (1:500) overnight at 4 °C. On the following day, the cells were washed with phosphate-buffered saline and incubated for 2 h in darkness at room temperature with Cy3-conjugated anti-rabbit secondary antibody . The cells were then washed thoroughly with phosphate-buffered saline and incubated overnight at 4 °C with Alexa 488-conjugated rabbit polyclonal anti-RAMP1, RAMP2, or RAMP3 antibody  endogenously express all three RAMP proteins [siRNAs used: RAMP1, sc-40894; RAMP2, sc-3678; RAMP3, sc-40896; Santa Cruz Biotechnology; For the evaluation of CALCRL expression in various human tumour entities, 290 completely anonymised, archived, formalin-fixed, and paraffin-embedded tumour specimens from 290 patients  granted permission for this retrospective analysis. For this type of study involving completely anonymised human specimens that had been archived for >10 years, for which clinical data were not obtained, formal patient consent was not required.For the evaluation of CALCRL expression in rat and mouse tissues, formalin-fixed and paraffin-embedded specimens from normal rat and mouse brain, pituitary, dorsal root ganglia, thymus, spleen, lymph nodes, lung, heart, liver, duodenum, jejunum, ileum, colon, pancreas, kidneys, adrenals, and testicles  were obtained from the archives of the Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.2O2 in methanol for 45 min. Subsequently, sections were microwaved in 10 mM citric acid (pH 6.0) for 16 min at 600 W.From the paraffin-embedded tissue specimens, 4-µm-thick sections were prepared and floated onto positively charged slides. After the sections were dried at room temperature, they were deparaffinised and rehydrated through a graded ethanol series. Endogenous peroxidases were then blocked by incubation in 0.3% HSingle-labelling immunohistochemistry was conducted by an indirect peroxidase labelling method. Sections were first incubated with the anti-CALCRL antibody 8H9L8 (1:500 dilution) overnight at 4 °C, then incubated with biotinylated anti-rabbit IgG and peroxidase-conjugated avidin  for 30 min each at room temperature. The chromogen comprised 3-amino-9-ethylcarbazole in acetate buffer . Sections were counterstained with Mayer’s haematoxylin and mounted in Vectamount™ mounting medium (Vector Laboratories). To establish controls for immunohistochemical analyses, the anti-CALCRL antibody 8H9L8 was either omitted, replaced by a rabbit IgG isotype control , or adsorbed for 2 h at room temperature with 10 µg/mL of the immunising peptide (peptide 2) or a control peptide that corresponded to a different region of the receptor  overnight at 4 °C. The sections were then washed with phosphate-buffered saline/1% bovine serum albumin and incubated for 2.5 h in darkness at room temperature with Cy3-conjugated anti-rabbit secondary antibody . After sections had been thoroughly washed with phosphate-buffered saline/1% bovine serum albumin, they were incubated overnight at 4 °C with the Alexa 488-conjugated rabbit polyclonal anti-RAMP1, -RAMP2, or -RAMP3 antibody . Finally, sections were mounted  and evaluated using an LSM 510 META laser scanning confocal microscope ; 488 nm (Alexa-488); 543 nm (Cy3)).The results of CALCRL single-labelling staining with the chromogen 3-amino-9-ethylcarbazole in human tumour specimens were scored using the semiquantitative Immunoreactivity Score (IRS) described by Remmele and Stegner (1987) . The perWe have generated and characterised a novel rabbit monoclonal anti-human CALCRL antibody that is well suited for visualising CALCRL expression in formalin-fixed, paraffin-embedded human, rat, and mouse tissues. This antibody is also suitable for Western blot analyses and immunocytochemistry experiments. To our knowledge, this antibody has facilitated the establishment of the first broad profile of CALCRL protein expression in diverse normal and neoplastic tissues from humans, as well as normal tissues from rats and mice. In addition to the confirmation of previous findings, this antibody enabled us to provide the first descriptions of CALCRL expression in many tumour entities. Among the tumour types investigated in this study, CALCRL was predominantly expressed in all types of thyroid carcinomas, as well as parathyroid adenomas, small-cell lung cancers, large cell neuroendocrine carcinomas of the lung, pancreatic neuroendocrine neoplasms, renal clear cell carcinomas, pheochromocytomas, lymphomas, and melanomas. In these tumours, CALCRL may represent a useful target structure for future therapies.


JAK2, PRDM16, LRP1B, NIN, and NKX2-1 genes) were supported by repeated testing on an independent set of Singaporean NPC patients and unaffected Singaporean controls. A FANCE variant was observed in two siblings with NPC, but not in three unaffected siblings of the same family. Gene-based burden testing recapitulated the association between NKX2-1 and FANCE variants with NPC risk. Pathway analysis revealed a higher frequency of germline mutations in endocytosis and immune-modulating pathways. Our research has identified new variants and genes associated with susceptibility to NPC, which are relevant for an improved understanding of the genetic predisposition of NPC.The aim of this study was to identify the germline genetic variants associated with an increased risk of developing nasopharyngeal carcinoma (NPC). DNA samples from 119 Singaporean NPC patients were sequenced, with 17 pathogenic variants in 17 genes found to be enriched in NPC patients as compared to unaffected controls. Five of these variants  from an independent case-control comparison of 156 NPC patients and 9770 unaffected individuals. In a family with five siblings, a FANCE variant (p. P445S) was detected in two affected members, but not in three unaffected members. Gene-based burden testing recapitulated variants in NKX2-1 and FANCE as being associated with NPC risk. Using pathway analysis, endocytosis and immune-modulating pathways were found to be enriched for mutation burden. This study has identified NPC-predisposing variants and genes which could shed new insights into the genetic predisposition of NPC.The current understanding of genetic susceptibility factors for nasopharyngeal carcinoma (NPC) is still incomplete. To identify novel germline variants associated with NPC predisposition, we analysed whole-exome sequencing data from 119 NPC patients from Singapore with a family history of NPC and/or with early-onset NPC, together with 1337 Singaporean participants without NPC. Variants were prioritised and filtered by selecting variants with minor allele frequencies of <1% in both local control (n = 1337) and gnomAD non-cancer (EAS) (n = 9626) cohorts and a high pathogenicity prediction (CADD score > 20). Using single-variant testing, we identified 17 rare pathogenic variants in 17 genes that were associated with NPC. Consistent evidence of enrichment in NPC patients was observed for five of these variants (in  Nasopharyngeal carcinoma (NPC) afflicted 129,000 new patients globally in 2018 . RecentlCRIP2 and MIPOL1, EBV oncoproteins LMP1 and LMP2, and increased EBV-associated tumorigenesis via specific EBV-infection-prone HLA haplotypes [NF-kB pathway and MLL3 gene [MST1R and RPA1 with a genetic predisposition for NPC and aggressive disease, respectively [CDKN2A/2B, BRD2, TNRFRSF19, and CLPTM1L/TERT [Potential genetic factors for NPC based on various models of tumorigenesis have implicated developmental genes such as plotypes . More reLL3 gene ,12,13,14ectively ,16. In aectively , and tarM1L/TERT .FANCE, JAK2, PRDM16, LRP1B, NIN, and NKX2-1 that may be implicated in the pathogenesis of NPC. Pathway analysis revealed that the endocytosis and immune-modulating pathways were enriched for mutation burden.Here, we performed WES on germline DNA from 119 NPC patients from Singapore, to determine the prevalence of mutations in previously reported NPC-associated genes and identify new NPC susceptibility variants. The variants identified in this cohort were also examined in an independent cohort of young 156 NPC cases who were diagnosed before they reached 40 years old. In addition, case-control association analyses were performed against local control and gnomAD non-cancer East Asian control cohorts. By using a stringent filtering and prioritisation strategy, we identified germline variants in Blood samples for the discovery cohort were obtained from 119 patients who were diagnosed with NPC from the National University Hospital, Singapore . These pFor the validation cohort, blood samples were obtained from 156 patients diagnosed with NPC at or below 40 years of age from the National Cancer Centre Singapore . UnaffecFor the discovery cohort and their family members, genomic DNA was extracted from peripheral blood mononuclear cells using routine laboratory methods . SequencFor each sequenced sample, read pairs were aligned to the human reference genome (b37) using BWA-MEM (v0.7.17) . The reaVariants were annotated with ANNOVAR , includiPotential NPC variants mutated in two or more affected patients were first selected from the jointly genotyped variants. Rare variants were selected by filtering for variants with minor allele frequency (MAF) less than 1% in both the gnomAD non-cancer (EAS) and the local control cohorts. Then, pathogenic variants were selected from nonsynonymous variants with CADD v1.3 phred score greater than 20. This stringent CADD threshold represents the top 1% of CADD-predicted pathogenic variants. Loss-of-function variants  were retained. Finally, rare pathogenic variants in known cancer genes were selected by choosing variants in genes appearing in at least two of the following cancer gene databases or literature sources: Network of Cancer Genes (NCG) 6.0 , COSMIC p-value less than 0.10) in both comparisons were selected.Principal component analysis (PCA) was performed to verify that participants with NPC and unaffected local control participants are of a similar genetic ancestry . PCA wasCase-control association analysis was repeated using a validation cohort of 156 NPC patients from Singapore, and the SG10K_Health control cohort (release 5.3) comprising of 9770 Chinese, Malay, and Indian healthy volunteers from Singapore .Germline variants from family members of discovery cohort probands were called using DRAGEN v3.8.4 with hg38 as reference genome [R2, and the number of variants to be filtered   for enriched pathways. Enrichment p-values for canonical pathways were calculated using the right-tailed Fisher’s Exact Test.The gene-based burden test on discovery and local controls was repeated with the same parameters, without filtering for known cancer genes. Then, 559 genes with Low-quality variants in both variant- and gene-based results were identified by checking their alignments in IGV  Supplem. For thep-values were corrected for multiple testing to reduce using the Benjamini–Hochberg method to reduce the false discovery rate [Variant-based case-control analyses were performed using a two-tailed Fisher’s Exact Test . Gene-baery rate .In the discovery cohort, patients either had an age at diagnosis of 40 years of age or younger (53/119 or 58.9%), first- or second-degree family history of any cancer (29/119 or 24.4%), or both (37/119 or 31.1%). In the validation cohort, all patients had an age at diagnosis of 40 years of age or younger. Both cohorts were predominantly ethnic Chinese: the discovery cohort comprised 105 ethnic Chinese (88.2%), 3 ethnic Malays (2.5%), 1 ethnic Indian (0.8%), and 10 NPC patients (8.4%) of other ethnicities; the validation cohort had 132 ethnic Chinese (84.6%), 9 ethnic Malays (5.8%), and 15 patients (9.6%) of other ethnicities .The 119 discovery cohort patients and 1337 local controls were jointly genotyped using GATK . In totaA list of singleton variants, each present only in one case, are shown in JAK2, PRDM16, BMPR1A and tumor-suppressor genes KMT2C, FAT4, and LRP1B [We performed case-control association analysis on 188 rare pathogenic variants in known cancer genes, comparing their allele frequencies in our discovery case cohort versus both local controls and gnomAD (EAS). Of these 188 variants, we shortlisted 17 variants, all of which were non-synonymous SNVs with a CADD PHRED score greater than 20, in 17 cancer-associated genes with substantially higher allele frequencies in patients with NPC as compared to both local control and gnomAD cohorts . These ind LRP1B   and healthy controls from SG10K_Health (n = 9770). Five of the 17 variants, in controls .NKX2-1 and NPC risk, as two of 119 cases (1.7%) but none of the 1337 controls had variants in NKX2-1 (FDR-adjusted p = 0.0144)  .FANCE had a significantly larger germline mutation burden, where two affected individuals (11.1%) but no unaffected individuals had a FANCE variant (rs141551053)  . The twoJAK2, PRDM16, LRP1B, NIN, and NKX2-1 with variants repeatedly enriched in variant-based analysis, and, FANCE, enriched in gene-based burden testing, using the TCGA database . Immune-modulating pathways were also significantly enriched, particularly GM-CSF signalling, “JAK1 and JAK3 in γc cytokine signalling”, and IL-15 production pathways .We performed pathway analysis using significant genes in a gene-based burden test of variants between discovery case and local control cohorts. The top ten canonical pathways based on the significance of enrichment p-values were also calculated for NPC patients and controls with variants in any genes in each of the implicated pathways. Our results show that all ten pathways were significantly enriched in our dataset  (Odds ratios and  < 0.05) .GABBR1, encoding a subunit of the GABA receptor, which were previously associated with NPC, were replicated in our cohort at p = 0.05 [BRD2 [BRD2 did not satisfy the criterion of being a known cancer gene in cancer gene databases or the literature sources  [We were able to replicate the results of six variants and four genes previously implicated in NPC by previous studies ,49,50,51p = 0.05 ,47. We a05 [BRD2 ,48, thou sources . Four ge 0.0394) ,15,18  were also associated with NPC in the validation case cohort.To identify novel germline variants predisposing one to NPC, we first examined WES of 119 Singaporean patients who had early-onset NPC and/or a family history of NPC, followed by an independent set of 156 early-onset NPC patients. Here, we discovered an initial list of 17 unique variants in 17 genes associated with NPC, five of which (in BMPR1A nonsynonymous SNV (rs55932635) was identified in one of 56 BRCA-negative breast cancer patients in Puerto Rico [JAK2 nonsynonymous SNV (rs200018153) was found in one of 1487 acute myeloid leukaemia (AML) patients in the United States [Of these 17 variants, two variants were previously reported in cancer-related studies. The rto Rico , while trs2000181 was founAPOB variant was absent from all three control cohorts  was detected in two affected siblings but not in three unaffected siblings. Furthermore, gene-based burden testing on 16 probands and 38 family members also showed an association between the FANCE gene and NPC. FANCE encodes a critical subunit of the Fanconi Anaemia (FA) nuclear complex [Notably, in a family with five siblings, the  complex , which f complex . While hSNV rs1411053 was NKX2-1 was identified from both variant-based case-control association analysis and gene-based burden testing of the discovery cohort, suggesting that it is associated with NPC predisposition. NKX2-1 is a homeobox transcription factor expressed in the adult thyroid, lung, bronchus, and nasopharynx [opharynx . In lungopharynx . Althougopharynx ,59,60,61opharynx . IKKβ isopharynx .JAK2 nonsynonymous SNV c.1174G>A (rs200018153) that was enriched in our discovery cohort. This variant was also detected in an independent validation cohort but did not reach statistical significance. Nonetheless, this JAK2 variant had the highest allele frequency of all our 17 variants, in both discovery and validation cohorts. JAK2 is a non-receptor tyrosine kinase, and plays an important role in regulating the JAK/STAT signalling pathway that controls cell proliferation, differentiation, survival, and cytokine-mediated immune responses [JAK2, which lead to the hyperactivation of the JAK/STAT pathway, have been observed in many cancer types [JAK2 mutation is JAK2-V617F, which has been reported to be associated with predisposition to myeloproliferative disorders [JAK2 somatic mutations found in exon 12, R683 and T875, have also been linked to hematological malignancies [JAK2 mutations in NPC, two research groups have identified amplifications in the JAK2 gene that are responsible for promoting cell proliferation and cell signalling in NPC [Finally, we identified a esponses . Mutatioer types ,65,66. Tisorders ,68. Othegnancies ,70,71. Ag in NPC ,72. Moreg in NPC .There is conflicting evidence on whether the cellular entry of EBV is facilitated by clathrin-mediated or caveolar-mediated endocytosis, or both ,45. In oGABBR1 and HLA-F, where such polymorphisms correlated with NPC risk [RPA1 (rs1131636) conferring tumor progression and therapeutic resistance in NPC, which ultimately affects the patient’s survival [BRCA2 germline alterations related to homologous recombination deficiency were found to be associated with poor clinical outcome in patients with NPC [MLL3 (KMT2C) in three family members of Italian descent [BRD2, a chromatin remodelling gene, in Taiwanese NPC families and sporadic NPC cases [MST1R as a candidate susceptibility gene for NPC [MST1R, Dai and colleagues from Hong Kong identified several candidate NPC-susceptibility genes, including TRMT10B. In this study, we evaluated the prevalence of these variants and genes in our NPC discovery cohort, replicating the association between NPC and six variants in BRD2 and GABBR1, and four genes .In recent years, various genomic approaches have been used to interrogate the genetic landscape of NPC susceptibility. For example, SNP genotyping studies have reported germline polymorphisms in the MHC class I and nearby genes such as NPC risk ,47. A moPA1 rs11336 conferwith NPC . Recentl descent . AnotherPC cases . Further for NPC ,77. Alonp < 0.05), perhaps due to an insufficient sample size. Additional studies in larger cohorts of different genetic ancestry and meta-analyses are required to assess the frequency and importance of these NPC susceptibility variants.As a study of rare variants, our analysis is limited by its sample size. This study had a discovery cohort of 119 NPC cases, and a validation cohort of 156 cases. Further, larger association studies will be necessary to detect rarer variants with greater certainty. For example, some of the variants and genes from previous NPC studies were present in our cohorts but failed to reach statistical significance (JAK2, PRDM16, LRP1B, NIN, NKX2-1 and FANCE were further supported by at least one of four additional tests: an additional variant-based case-control association analysis on an independent validation cohort, gene-based testing on the original discovery cohort; gene-based testing and co-segregation analysis of family members of the discovery cohort. Our study provides new insights into the genetic susceptibility to NPC, which will facilitate further investigations with the potential to translate the findings into future clinical practice. This warrants further functional characterization of the FANCE, NKX2-1, and JAK2 variants and elucidation of the mechanisms for the role of these genes in NPC development.In summary, we identified 17 germline variants in 17 genes that are associated with NPC predisposition. Six of these variants in six genes


We tested whether a model identifying prostate cancer (PCa) patients at risk of pT3-4/pN1 can be developed for use during COVID19 pandemic, in order to guarantee appropriate treatment to patients harboring advanced disease patients without compromising sustainability of care delivery.Within the Surveillance, Epidemiology and End Results database 2010-2016, we identified 27,529 patients with localized PCa and treated with radical prostatectomy. A multivariable logistic regression model predicting presence of pT3-4/pN1 disease was fitted within a development cohort . Subsequently, external validation  and head-to-head comparison with NCCN risk group stratification was performed.In model development, age, PSA, biopsy Gleason Grade Group (GGG) and percentage of positive biopsy cores were independent predictors of pT3-4/pN1 stage. In external validation, prediction of pT3-4/pN1 with novel nomogram was 74% accurate versus 68% for NCCN risk group stratification. Nomogram achieved better calibration and showed net-benefit over NCCN risk group stratification in decision curve analyses. The use of nomogram cut-off of 49% resulted in pT3-4/pN1 rate of 65%, instead of the average 35%.The newly developed, externally validated nomogram predicts presence of pT3-4/pN1 better than NCCN risk group stratification and allows to focus radical prostatectomy treatment on individuals at highest risk of pT3-4/pN1. Extraordinary demands are placed on healthcare system during the COVID19 pandemic. In consequence, non-COVID19 related activities are curbed . RadicalNCCN guidelines stratify prostate cancer (PCa) patients into very low, low, favorable intermediate, unfavorable intermediate, high and very high risk groups according to their clinical parameters, such as PSA levels, clinical T stage, Gleason Grade Group at biopsy, number and percentage of positive biopsy cores. The optimal therapeutic strategy differs among groups, ranging from active surveillance for very low and low risk patients, to surgery or radiotherapy for the other groups . The ratIn the Surveillance, Epidemiology and End Results (SEER) database , we focuThe endpoint of interest consisted of the ability to identify patients with extra-prostatic disease at RP, defined as pT3-4/pN1, using clinical characteristics: age at diagnosis, PSA, clinical T stage, biopsy GGG and percentage of positive biopsy cores.Mean and standard deviations were reported for normally distributed, continuously coded variables. Median and interquartile ranges were reported for non-normally distributed, continuously coded variables. Frequencies and proportions were reported for categorical variables. The t-test, Mann-Whitney and chi-squared tests were used to compare means, medians and proportions, respectively. Statistical analyses consisted of several steps.First, within the development cohort  we fitted a logistic regression model predicting pT3-4/pN1 using age at diagnosis, PSA level, biopsy GGG and percentage of positive biopsy cores. Clinical T stage was not included in the final model, due to lack of statistical significance. Moreover, based on left-skewed distribution of PSA level, cubic spline-transformed values were used in nomogram development.Second, within the external validation cohort , we tested the discriminant ability of the newly developed nomogram, as well as that of the NCCN risk group stratification. Testing relied on ROC-derived area under the curve (AUC) that assessed the discriminant ability of nomogram versus the NCCN risk group stratification. Statistical significance of differences in related AUC values was tested according to DeLong et al. methodology . Moreovewww.r-project.org)  . All tesWithin the SEER database, we identified 27,529 patients that harbored favorable intermediate, unfavorable intermediate, high risk or very high risk localized PCa, according to NCCN risk group stratification . Of thosWithin the development cohort, the multivariable logistic regression model underlying the nomogram predicting the probability of pT3-4/pN1 rested on age, PSA level, biopsy GGG and percentage of positive biopsy cores Figure 1Within the external validation cohort, the nomogram yielded an AUC of 74.4% (95% CI 73.5 – 75.3%) versus 68.0% (95%CI 67.1 – 68.9%) for the NCCN risk group stratification (p<0.001). In calibration plots, comparisons between predicted and observed values yielded smaller departures from ideal predictions for the nomogram, relative to the NCCN risk group stratification. Specifically, for the nomogram departures from ideal predictions ranged from -0.9 to +1.2%, for five equally-sized groups. Conversely, for the NCCN risk groups departures from ideal predictions ranged from +0.1 to +2.9% Figure 2Finally, we tested specific nomogram cut-offs that corresponded to the predefined NCCN risk group levels. For example, a nomogram cut-off of 49% would identify 3,321 patients (24.5%) of the original 13,552 patients as at high risk of pT3-4/pN1. Of those individuals, 2,147 (64.6%) indeed harbored pT3-4/pN1. This cut-off virtually perfectly corresponded to the definition of high risk or higher, according to NCCN risk group stratification. This definition (high risk or very high risk) identified virtually the same number of individuals: 3,337 (24.6%) for NCCN versus 3,321 (24.5%) for the nomogram. Of those 3,337 individuals, 1,879 (56.3%) indeed harbored pT3-4/pN1, versus 2,147 out of 3,321 (64.6%) for the nomogram Table 2.During COVID19 pandemic RPs for localized intermediate or high risk PCa qualify for potential postponement up to 3 months or even beyond, according to guideline recommendations –13. HoweFirst, within the overall cohort of 27,529 patients with clinically localized intermediate or high risk PCa, 34% of patients harbored pT3-4/pN1 stage. The rate of pT3-4/pN1 ranged from 18.3 to 66.7% in respectively favorable intermediate (18.3%), unfavorable intermediate (36.6%), high (42.5%) and very high (66.7%) risk PCa patients. According to individual patient characteristics, the rate of pT3-4/pN1 demonstrated important variability, even within those four risk groups. In consequence, the individual risk of pT3-4/pN1 stage is highly variable and cannot be precisely ascertained with the use of NCCN risk group stratification alone. In consequence, accurate prediction of individual pT3-4/pN1 probability prior to RP represents an unmet need. Previous nomograms developed by this group of investigators  and by oSecond, to address the study objective, we relied on the development cohort of 13,977 patients residing in Midwestern, Southern and North-Eastern United States to identify independent predictors of pT3-4/pN1 at RP, within a multivariable logistic regression model. Those consisted of age, PSA level, biopsy GGG and percentage of positive biopsy cores. The logistic regression model was then graphically converted into nomogram format. Within that nomogram, biopsy GGG and percentage of positive biopsy cores contributed the highest possible number of risk points. Nonetheless, they were closely followed by PSA level and age. These observations indicate that patient, biochemical, as well as biopsy tumor characteristics represent important predictors of pT3-4/pN1 probability at RP. Moreover, in its graphical representation, the nomogram exhibits the importance of dynamic interactions between different levels of risk factors, regardless of their absolute values and across their entire range, without pre-defined cut-offs.Third, the application of the newly developed nomogram in the external validation cohort yielded several important results. First, its accuracy was higher than the one of NCCN risk group stratification (74 vs 68%). Second, its calibration revealed lesser departures from ideal predictions than those of NCCN risk group stratification. Finally in DCA, a higher net-benefit was recorded for the nomogram than for the NCCN risk group stratification. Taken together, the newly developed nomogram exhibited better performance than the NCCN risk group stratification, according to three classic testing benchmarks for a predictive tool.Fourth, although the nomogram can provide an individual probability of pT3-4/pN1, we relied on the use of cut-offs to illustrate its relative benefit versus NCCN risk group stratification. For example, the use of a 49% nomogram cut-off allowed us to identify 3,321 patients. Within those, 2,147 (64.6%) harbored pT3-4/pN1. The cut-off of 49% virtually perfectly replicated the NCCN definition of high risk PCa. The use of that definition also identified 3,337 patients. Of those, 1,879 (56.3%) harbored pT3-4/pN1. In consequence, despite virtually the same numbers of high risk individuals identified by the nomogram  versus NCCN risk group stratification , the rate of observed pT3-4/pN1 was higher for the nomogram than for the NCCN risk group stratification (64.6 vs 56.3%). In consequence, it may be concluded that the newly developed nomogram outperformed the NCCN risk group stratification based on three established statistical benchmarks. The use of nomogram cut-offs also yielded a higher proportion of individuals with pathologically confirmed pT3-4/pN1 than the NCCN risk group stratification. The same scenario was recorded when a lower nomogram cut-off (more sensitive) was compared to the NCCN risk group stratification, as well as when a higher nomogram cut-off (more specific) was compared to the NCCN risk group stratification.Several nomograms have already been proposed for PCa patients stratification , 27–30. Taken together, we developed a new nomogram to identify individuals with pathologically proven pT3-4/pN1 stage, in contemporary favorable intermediate, unfavorable intermediate, high and very high risk patients. In those PCa patients, the nomogram outperformed the existing NCCN risk group stratification, based on the three established testing benchmarks, namely accuracy, calibration and DCA. Finally, the application of nomogram cut-offs resulted in a higher proportion of individuals with pathologically proven pT3-4/pN1 stage than the use of corresponding NCCN risk group definitions. In consequence, the use the nomogram represents a better alternative to NCCN risk group stratification.Our study is not devoid of limitations, such as its retrospective and population-based nature that results in a limited number of assessable variables. For example, prostate magnetic resonance imaging findings, as well as molecular and genetic tests, repeat biopsy information or percentage of cancer per core were unavailable , 32. MorThe newly developed, externally validated nomogram predicts presence of pT3-4/pN1 better than NCCN risk group stratification and allows to focus radical prostatectomy treatment on individuals at highest risk of pT3-4/pN1, during COVID19 pandemic and similar crises.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.LN: conception and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, statistical analysis LS: acquisition of data, analysis and interpretation of data, critical revision of the manuscript CR: acquisition of data, analysis and interpretation of data, critical revision of the manuscript MW: acquisition of data, analysis and interpretation of data, critical revision of the manuscript CW: acquisition of data, analysis and interpretation of data, critical revision of the manuscript ZT: statistical analysis GG: critical revision of the manuscript NF: critical revision of the manuscript VM: critical revision of the manuscript FC: critical revision of the manuscript SS: critical revision of the manuscript MG: critical revision of the manuscript FS: critical revision of the manuscript FM: critical revision of the manuscript, supervision AB: critical revision of the manuscript, supervision PK: conception and design, drafting of the manuscript, critical revision of the manuscript, supervision. All authors contributed to the article and approved the submitted version.


Genypterus chilensis) is a native Chilean species important for aquaculture diversification in Chile. The effect of high-temperature stress on the liver, a key organ for fish metabolism, is unknown. In this study we determined for the first time the effects of high-temperature stress on the liver of red cusk-eel. The results showed that high-temperature stress increased hepatic enzyme activity in the plasma of stressed fish. Additionally, this stressor generated oxidative damage in liver, and generated a transcriptional response with 1239 down-regulated and 1339 up-regulated transcripts associated with several processes, including unfolded protein response, heat shock response and oxidative stress, among others. Together, these results indicate that high-temperature stress generates a relevant impact on liver, with should be considered for the aquaculture and fisheries industry of this species under a climate change scenario.The red cusk-eel (Genypterus chilensis), a native fish for which knowledge on environmental stressors effects is limited. This study evaluated the effects of high-temperature stress on the liver of red cusk-eel in control (14 °C) and high-temperature (19 °C) groups using multiple approaches: determination of plasmatic hepatic enzymes , oxidative damage evaluation , and RNA-seq analysis. High-temperature stress generated a significant increase in hepatic enzyme activity in plasma. In the liver, a transcriptional regulation was observed, with 1239 down-regulated and 1339 up-regulated transcripts. Additionally, high-temperature stress generated oxidative stress in the liver, with oxidative damage and transcriptional modulation of the antioxidant response. Furthermore, an unfolded protein response was observed, with several pathways enriched, as well as a heat shock response, with several heat shock proteins up regulated, suggesting candidate biomarkers  for thermal stress evaluation in this species. The present study shows that high-temperature stress generated a major effect on the liver of red cusk-eel, knowledge to consider for the aquaculture and fisheries of this species.Environmental stressors, such as temperature, are relevant factors that could generate a negative effect on several tissues in fish. A key fish species for Chilean aquaculture diversification is the red cusk-eel ( Environmental factors are important for the physiology of fish, particularly those associated with water conditions. Among these, temperature, pH, and dissolved oxygen (DO) are crucial for the homeostasis of the fish in marine environments, and changes in these factors could lead to generating a stressful status for the animal . It has Genypterus chilensis), part of the Genypterus genus, endemic to the South Pacific coast, an economically relevant fish for fisheries and, recently, part of the Chilean aquaculture diversification program [Chile is a relevant country in seafood production, with an important fishery and aquaculture industry. Additionally, the Chilean aquaculture sector is recognized for its important salmon and mussel industries . However program ,11, with program . The red program  and is c program . In thisG. chilensis showed a variable tissue response under handling stress, with altered metabolic status in the liver, a modulation of the immune response in the head kidney, and an induction of atrophy in skeletal muscle through coding and noncoding regulation [Genypterus species has not been previously studied, nor has the impact of this stressor on the oxidative status of this tissue. It is important to consider that sea temperatures will increase through sea heat waves due to the effect of ENSO under a climate change scenario, which will affect the Chilean coast associated with the geographic range of G. chilensis. Therefore, the objective of this study was to evaluate the effect of high-temperature stress on the liver of G. chilensis in terms of the transcriptomic and oxidative stress status to determine the negative impact of this type of stressor on liver.Stress in fish can be characterized as an adaptative response to danger, which generates physiological changes to prepare the fish to respond and survive threats. This stress response in fish is mediated by the hypothalamic–pituitary–interrenal (HPI) axis though several key chemical mediators, including corticotropin releasing factor, adrenocorticotropic hormone, α-melanocyte-stimulating hormone, adrenaline, and cortisol, a key hormone that increases in plasma under stress . If the gulation . Additiogulation , as wellgulation . One of gulation . NeverthAll procedures with the red cusk-eel individuals and all scientific activities adhered to animal welfare procedures and were approved by the bioethical committees of the Universidad Andres Bello (007/2018) and the National Commission for Scientific and Technological Research (CONICYT) of the Chilean government.G. chilensis) of 12 months of age , collected from the Centro de Investigación Marina de Quintay (CIMARQ), maintained under natural photoperiod conditions (L:D 12:12), and controlled temperature (14 ± 1 °C), and fed daily with commercial pellet food. Fish were separated into control and stress groups, with the stress group subjected to a standardized thermal stress protocol proven to generate stress in red cusk-eel [g for 10 min for serum obtention and stored at −80 °C until analysis. After blood sampling, fish were euthanized . Fish livers were collected and stored for RNA extraction in RNAsave solution  or immediately frozen in liquid nitrogen and stored at −80 °C until analysis for oxidative damage evaluation.In this study, we used reproductively immature red cusk-eel juveniles (cusk-eel . Brieflycusk-eel . The conThe plasmatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP) were determined using commercially available kits from Valtek  following the manufacturer’s instruction. Briefly, these kits determined the enzymatic activity (IU/L) via the generation of colorimetric products from glutamate (colorimetric product: 450 nm), pyruvate (535 nm), and p-nitrophenol (405 nm) for AST, ALT and AP activity, respectively.g centrifugation. Proteins were quantified using the Pierce BCA Protein Assay Kit . Then, carbonylated protein content was quantified using the OxiSelect Protein Carbonyl Spectrophotometric Assay  following the manufacturer’s protocol. The lipid peroxidation determination in the liver was performed using the OxiSelect HNE Adduct Competitive ELISA Kit , following the manufacturer’s protocol. This kit determines lipid peroxidation through the quantification of hydroxynonenal (HNE) protein adducts in the extracted proteins of the liver.To determine the oxidative damage in the liver of red cusk-eel in response to high-temperature stress, DNA oxidative damage, protein carbonylation, and lipid peroxidation were evaluated using commercially available kits. The DNA oxidative damage assay was performed with genomic DNA (gDNA) purified from 25 mg of the liver using the DNAzol reagent  following the manufacturer’s protocol and quantified with the Epoch Spectrophotometer System . Then, the apurinic/apyrimidinic sites (AP sites) were determined in the gDNA using the OxiSelect Oxidative DNA Damage Quantification Kit  following the manufacturer’s protocol. The protein carbonylation assay was performed using total protein extracted from 100 mg of the liver in 1 mL of lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40, solubilized at 4 °C after 12,000× ® reagent  protocol. Total RNA was quantified by fluorometry with the Qubit® RNA quantitation assay  and purity was determined according to 260/280 ratio using the Epoch Spectrophotometer System . The RNA integrity was measured according to RNA Quality Measurement Number (RQN) through a Fragment Analyzer with the Standard Sensitivity RNA Analysis kit , selecting samples with RQN ≥ 8. The cDNA libraries construction were performed with 1 µg of total RNA per sample using the Illumina® TruSeq RNA Sample Prep Kit v2 , following the manufacturer’s protocol. The sizes of the mRNA libraries were determined through a Fragment Analyzer using the NGS Fragment Analysis kit  and quantified by qPCR using the Kapa Library Quantification kit . Paired-end sequencing (2 × 100 bp) was performed on a Hiseq 2500 (Illumina®) platform in Macrogen Inc. .Total RNA was extracted from the liver stored in RNAsave solution  using the TRIzolG. chilensis reference transcriptome previously annotated by our group , using the CLC Genomics Workbench v.7.0.3 software. The filtered reads were mapped to a r group , following the manufacturer’s protocol. A total of thirteen differentially expressed transcripts were selected for qPCR validation, corresponding to: hsp60, hsp70, gpx7, ddit4, leptin, msh2, msh3, c1ql1, ccl20, atg12, atg4b, casp3 and c3. Primers were designed with Primer 3 software v0.4.0  using the reference transcriptome previously described. The primer sequences, amplicon size, Tm, and efficiency are presented in ® II SYBR® Green master mix , 100 ng of cDNA per reaction and 200 nM of each primer in a 15 μL final volume. Thermal cycling conditions were an initial activation of 2 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C. A melt curve analysis was performed to confirm a single qPCR product and a standard curve using two-fold series dilutions was used to estimate the efficiency of each primer set. The expression of target genes was normalized using the geometric means of two reference genes (actb and taf12) previously validated for red cusk-eel in the liver [Total RNA previously extracted was DNAse-treated to remove residual gDNA, and 1 μg of RNA was reverse transcribed into cDNA using the QuantiTecthe liver  and follhe liver .t-test with a significance threshold of p < 0.05. All statistical analyses were performed using GraphPad Prism, v.5.00 .Significant differences between means of the control and stress groups for DNA damage (AP sites), enzymatic activity  and differential expression of the qPCR validated genes were determined using a G. chilensis [We previously reported that thermal stress for 5 days significantly increased plasmatic levels of cortisol and glucose in hilensis . To undeG. chilensis, we performed RNA-seq analysis on the liver of each experimental group. The sequencing generated a total of 754,678,455 paired-end reads, with an average of 58,052,189 raw paired-end reads per library. The raw data are available from NCBI under BioProject PRJNA835467 with BioSamples accession number SAMN28102858, SAMN28102859, SAMN28102860, SAMN28102861, SAMN28102862, SAMN28102863, SAMN28102864, SAMN28102865, SAMN28102866, and SAMN28102867. After trimming by quality, adapters, and size, we obtained an average of 58,018,122 high-quality filtered paired-end reads per library (G. chilensis reference transcriptome (NCBI accession number SRS614525), obtaining an average of 85.2% mapped reads , heat shock protein 70 (hsp70) and heat shock protein 90 (hsp90).In the KEGG pathway analysis, we identified several pathways represented in the down-regulated and up-regulated transcripts, including apoptosis, cell cycle, MAPK signaling pathway, autophagy, protein processing in endoplasmic reticulum, and ubiquitin-mediated proteolysis. Among these pathways, protein processing in endoplasmic reticulum was one of the most represented on the differentially expressed transcripts , with 31hsp60), hsp70, DNA damage-inducible transcript 4 (ddit4), glutathione peroxidase 7-like (gpx7), leptin, DNA mismatch repair Msh2-like (msh2), DNA mismatch repair Msh3-like (msh3), complement C1q 2-like (clql1), C-C motif chemokine 20-like (ccl20), ubiquitin ATG12-like (atg12), cysteine protease ATG4B-like (atg4b), caspase 3 (casp3) and complement C3-like (c3)  . All of ike (c3) , with a ike (c3) .Genypterus species are limited [Genypterus species. In this sense, thermal stress has been previously studied in other tissues of Genypterus species, including the skeletal muscle, ovary, and post-ovulatory eggs [Stress in fish is a relevant issue for aquaculture species and native populations of fish . The env limited ,26,37,38ory eggs ,24. TherLabeo rohita) [Oreochromis niloticus) [Channa punctatus) [Takifugu obscurus) [Scophthalmus maximus) [In a previous study, we determined that high temperature (19 °C) could generate a stress response in red cusk-eel with an increase in the plasmatic level of cortisol and glucose . In the  rohita) ,40, metaloticus) ,42 and snctatus) , and elenctatus) . In termbscurus) , which imaximus)  under himaximus) .Chanos Chanos) [Piaractus mesopotamicus) under low-temperature stress [Solea senegalensis) [Labeo rohita), with lipid peroxidation and DNA fragmentation [Acanthopagrus schlegeli) [gpx1 was increased, an effect not observed for this species in skeletal muscle [msh2 and msh3), concordant with the previously reported effect of thermal stress on zebrafish (Danio rerio) [Cyprinodon variegatus), where a limited effect on antioxidant enzymes and no lipid peroxidation were present [Oxidative stress corresponds to a disturbance between the production of ROS, which can accumulate in cells, and the antioxidant defenses generated by the cellular systems to detoxify these ROS . OS part Chanos) , as welle stress ,54. Highalensis) . Additioentation . This ishlegeli)  and puffhlegeli) , as wellhlegeli) , where gl muscle . Additioo rerio)  and Amero rerio) . However present , in cont present . We prev present , but no  present , showingGenypterus genus. We observed a higher hepatic transcriptional response to high-temperature stress compared to other types of stressors, such as handling stress, previously observed for red cusk-eel  [srp14 and srp19) and signal peptidase complex subunit 2-like (spcs2) [traf2), and mainly an up-regulation of this pathway, including ubiquilin-4 (ubqln4), cytoskeleton-associated protein 4 (ckap4); ribosome-binding protein 1 ; and glucosidase 2 subunit beta (prkcsh). Others include transcripts associated with the unfolded protein response, including eukaryotic translation initiation factor 2 alpha kinase 1 , 3  and 5 (eik2ak5), cyclic AMP-dependent transcription factor ATF-4-like (atf4) and DNA damage-inducible transcript 3 -like . The unfolded protein response could be initiated by EIK2AK3 kinase activation of eIF2α, leading to ribosome inhibition and attenuating protein synthesis [Colossoma macropomum) [Sparus aurata) [Temperature can modulate several cellular processes through gene expression regulation . Here, wscripts) , evidencscripts) . Howeverscripts) . This ER (spcs2) ; transcrynthesis . Additioynthesis , evidencynthesis ,68 and fropomum)  and gilt aurata) .hsp40, hsp60, hsp70, hsp90, and serpinh1. The up-regulation of these Hsps was previously observed in other fish under thermal stress, with an increase in the liver of three-spined stickleback (Gasterosteus aculeatus)  [Salmo salar) (hsp70) [Gadus morhua) (hsp70 and hsp90) [Megalobrama amblycephala)  [hsp60 and hsp70 expression levels were also regulated under thermal stress in the postovulatory eggs and skeletal muscle of red cusk-eel [serpinh1  is a chaperone involved in the biosynthesis of collagen at the ER [Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha) [sherpinh1 is one of the most suitable transcriptional biomarkers of high-temperature stress in fish. This was also the case for red cusk-eel in this study, representing a useful tool to evaluate thermal stress status in this species under a climate change scenario.The cellular response to thermal stress is a key process to preserve protein integrity; this is known as the heat shock response, which includes heat shock proteins (Hsps) to re-fold the proteins damaged by temperature . This red hsp90) , Atlanti (hsp70) ,74, Atlad hsp90) . The hspcusk-eel ,24, showt the ER , with a t the ER . It was wytscha) ,77, as wwytscha) , evidencG. chilensis, using a multiple approach of plasmatic hepatic enzymes, oxidative damage evaluation and RNA-seq analysis. We showed that high-temperature stress under heatwaves ENSO-associated scenario generated a major effect in the liver, affecting hepatic enzymes, generating oxidative damage in this tissue, as well as generating an unfolded protein response at the molecular level in several associated pathways, including a heat shock response, evidencing the affection of red cusk-eel under this type of stressor. This study contributes to knowledge about thermal stress under a climate change scenario, generating candidate biomarkers for thermal stress evaluation in this species, information that should be relevant for the aquaculture and fisheries industry of red cusk-eel.The present study evaluated for the first time the effects of high-temperature stress in the liver of


Ischemic stroke (IS) is a common neurological disorder associated with high disability rates and mortality rates. At present, recombinant tissue plasminogen activator (r-tPA) is the only US(FDA)-approved drug for IS. However, due to the narrow therapeutic window and risk of intracerebral hemorrhage, r-tPA is currently used in less than 5% of stroke patients. Natural compounds have been widely used in the treatment of IS in China and have a wide range of therapeutic effects on IS by regulating multiple targets and signaling pathways. The keywords “ischemia stroke, traditional Chinese Medicine, Chinese herbal medicine, natural compounds” were used to search the relevant literature in PubMed and other databases over the past five years. The results showed that JAK/STAT, NF-κB, MAPK, Notch, Nrf2, and PI3K/Akt are the key pathways, and SIRT1, MMP9, TLR4, HIF-α are the key targets for the natural compounds from traditional Chinese medicine in treating IS. This study aims to update and summarize the signaling pathways and targets of natural compounds in the treatment of IS, and provide a base of information for the future development of effective treatments for IS. Stroke is associated with the second leading cause of death and the third leading cause of disability among human diseases , and IS IS is a complex pathological cascade reaction involving various pathological factors, including oxidative stress, inflammation, apoptosis, autophagy, and BBB damage. Oxidative stress, inflammation, and apoptosis are the critical factors in cerebral ischemic injury ,9. An imTraditional Chinese medicine (TCM) has been practiced in China for thousands of years, and has gained wide clinical application , numerouThe JAK/STAT signaling pathway is involved in various physiological processes, such as cell proliferation, differentiation, and apoptosis. The JAK protein tyrosine kinase family consists of JAKl, JAK2, JAK3, and Tyk2. To date, seven members of the STAT family have been ascertained: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 . ExtraceSophora flavescens Aiton., has been shown to reduce the expression of the p-JAK2 and p-STAT3 proteins and the number of apoptotic cells in the brain tissue of Middle Cerebral Artery Occlusion (MCAO) rats, and plays a neuroprotective role by inhibiting the activation of JAK-STAT signaling pathway and reducing the inflammatory response [Carthamus tinctorius L., it can significantly down-regulate the expression of JAK2-mediated signaling due to ischemic injury, while significantly promoting the expression of SOCS3, which is a negative regulator of STAT3. By modulating the cross between JAK2/STAT3, Hydroxy saffron yellow A can confer neuroprotection against focal cerebral ischemia [Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey., has multiple pharmacological activities, it can increase blood flow in ischemic brain tissues of MCAO rats, upregulate EPO and EPOR expression, promote STAT3 phosphorylation and inhibit VEGF mRNA expression, thus improving blood supply to ischemic brain tissues, reducing vascular permeability and promoting angiogenesis through the JAK2/STAT3 signaling pathway [Carthamus tinctorius L., can increase the protein expression level of Bcl-2 and downregulate the expression of p-JAK2, p-STAT3, caspase-3 and Bax, and inhibit the JAK2/STAT3 signaling pathway to alleviate apoptosis caused by cerebral ischemia-reperfusion injury (CIRI) [Matrine, an alkaloid that is extracted from response . Hydroxyischemia . Catalpo pathway . Nicotify (CIRI) . Additioy (CIRI) ,26. The The NF-κB signaling pathway is a classic signal transduction pathway mediated by cytokines. It plays an important role in several physiological and pathological activities, including inflammation, oxidative stress, endothelial cell injury, and cell death . NF-κB iScutellaria baicalensis Georgi, exerts protective effects against cerebral ischemia by inhibiting TLR4/NF-ĸB signaling pathway and suppressing mitochondrial apoptosis, inflammation, and oxidative stress [Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., can promote FOXO3a phosphorylation and inhibit NF-κB nuclear translocation, which in turn reduces the levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and MPO, and exerts a protective effect against ischemic brain injury by reducing the inflammatory response through the FOXO3a/NF-κB signaling pathway [Schisandra chinensis (Turcz.) Baill., can inhibit TLR4 expression and NF-κB activation and reduces TNF-α, IL-6 and IL-1β levels, exerts protective effects against cerebral ischemia by inhibiting TLR4/NF-κB signaling pathway [Ephedra sinica Stapf, has been shown to decrease oxidative stress, prevent inflammation, increase immunological function, and decrease CIRI, which may be due to their suppression of NF-κB-NLRP3 signaling [Salvia miltiorrhiza Bunge, inhibits NF-κB activation and inflammatory factor release mediated by HMGB1-TLR4 signaling and attenuates HMGB1-mediated inflammatory response by inhibiting TLR4/MyD88/NF-κB signaling pathway [Artesunate, a derivative of artemisinin, reduces tissue damage caused by traumatic brain injury and protects MCAO mice from inflammatory injury by inhibiting NF-κB, releasing the pro-inflammatory cytokines IL-1β and TNF-α, reducing neutrophil infiltration, and inhibiting microglia activation . Skullcae stress . Syringi pathway . Schisan pathway . Ephedriignaling . Salvian pathway . Furthor pathway ,42,43,44Recent research demonstrated that the MAPK pathway plays an essential role in the initiation and progression of IS . The MAPCitrus reticulata Blanco, can reduce ischaemic/reperfusion-induced brain apoptosis by upregulating Bcl-2 expression, downregulating Bax and caspase-3 expression, and reducing the levels of pro-inflammatory factors TNF-α and IL-6 and the expression of p-p38 and MAPAP-2 in MCAO rats. This mechanism is related to the MAPK signaling pathway [Rheum palmatum L., can induce the expression of Bcl-2 and GLT-1 through the ERK-1/2 signaling pathway, inhibits neuronal apoptosis and ROS production, reduces glutamate toxicity, and alleviates nerve cell injury in a rat model of MCAO [Nobiletin, a flavonoid extracted from  pathway . Coriolu pathway . Scrophu pathway . Emodin, of MCAO . Further of MCAO ,57,58. TNotch is a highly conservative signaling pathway that plays a critical role in cell proliferation, differentiation, and apoptosis , it is aAstragalus penduliflorus subsp. mongholicus (Bunge) X. Y. Zhu significantly reduced the infarct area in MCAO rats, and promoted cell proliferation and duct formation, which in turn promoted angiogenesis and had a protective effect against cerebral ischemic injury, which was closely related to the upregulation of miRNA-210 expression, induction of HIF-VEGF-Notch signaling pathway activation and inhibition of target gene ewitinA3 expression [Cnidium monnieri (L.) Cusson., can significantly reduce the volume of cerebral infarction, reduce apoptosis, increase the expression of target proteins Notch1, Hes-5 and NICD by acting on the Notch pathway, and play a protective role for neurons [Astragaloside IV, the main saponin isolated from roots of pression . Osthole neurons . The NotThe Nrf2 signaling pathway plays a significant role in the occurrence and development of IS, and it can regulate the ability of cells to resist oxidative stress and protect brain tissue . Nrf2 beTrifolium pratense L., promotes the nuclear translocation of Nrf2 and induces the expression of HO-1 by regulating the Nrf2/HO-1 signaling pathway, it protects the rat brain from ischemic injury through antioxidant and anti-inflammatory effects [Coptis chinensis Franch., can reduce oxidative stress, inflammatory response, and neuronal apoptosis in MCAO mice by activating the AMPK/ Nrf2 pathway [Taraxacum mongolicum Hand.-Mazz., can significantly inhibit the generation of ROS and MDA in hippocampal neurons induced by OGD/R, leading to a decrease in caspase-3 and Bcl-2 expression, and a concurrent increase in the expression of Bax, HO-1, NQO-1, and GPX-3. Taraxasterol can protect hippocampal neurons from OGD/R-induced injury by activating the Nrf2 signaling pathway [Biochanin A, the main flavonoid component of  effects . Rosmari effects , can up- effects . Adenosi pathway . Taraxas pathway . In addi pathway ,77. The There are many experimental studies on the regulatory role of the PI3K/Akt signaling pathway in IS ,79,80. TReynoutria japonica Houtt. and Vitis vinifera L., it reduces the expression of IL-1β, COX-2 and TNF-α by stimulating the PI3K/Akt signaling pathway as well as decreasing infiltration of neutrophils, thereby reducing the inflammatory response in rats with ischemic stroke [Ligusticum chuanxiong Hort., can significantly increase the levels of p-Akt and p-eNOS in the brain tissue of MCAO rats, and play a neuroprotective role on the brain of ischaemic/reperfusion injury rats by stimulating the PI3K/Akt pathway [Polygala tenuifolia Willd., can downregulate the expression of Bcl-2/Bax and caspase-3 in PC12 cells and prevent OGD/R-induced injury by stimulating the PI3K/Akt signaling pathway [Puerariae Lobata (Willd.) Ohwi, can significantly increase the expression of Akt1, GSK-3β, and MCL-1 p62 as well as decrease caspase-3 expression levels in MCAO rats. These findings indicate that puerarin can regulate the neuroprotective mechanism of autophagy via the PI3K/Akt1/GSK-3β/MCL-1 signaling pathway [Resveratrol is a natural polyphenol isolated from plants such as c stroke . Ligustr pathway . Polygal pathway . Puerari pathway . In addi pathway ,92. The Magnolia officinalis Rehd. Et Wils) and Salvianolic acid B  can regulate brain injury induced by cerebral ischemia by activating SIRT1, deacetylating to inhibit Ac-FOXO1 expression, and suppressing inflammatory cytokines and apoptosis [O-β-D-glucoside, a flavonoid isolated from Astragalus penduliflorus subsp. mongholicus var. dahuricus (Fisch. ex DC.) X. Y. Zhu, can attenuate OGD/R-induced oxidative stress and neuronal apoptosis by activating SIRT1 and upregulating FOXO1 and PGC-1 α expression [SIRT1 is a nicotinamide adenine dinucleotide-dependent histone deacetylase with deacetylation of various histones and non-histones , which cpoptosis ,98. Calypression . MoreoveEpimedium brevicornu Maxim.) and ursolic acid  R. J. Wang and Actinidia chinensis Planch.) could further inhibit neuronal apoptosis by regulating the balance of MMP9/TIMP1, thereby significantly improving the ischemia-reperfusion induced BBB disruption in MCAO rats, preventing cerebral ischemia-reperfusion injury [Astragalus penduliflorus subsp. mongholicus var. dahuricus (Fisch. ex DC.) X. Y. Zhu) and oxymatrine  can reduce the expression of MMP9 protein by downregulating the expression of CAV1, thereby improving the integrity of the BBB after CIRI [MMP9 is a member of the zinc-dependent protein hydrolase family and can degrade extracellular matrix, including collagen IV, laminin, and fibronectin . MMP9 exn injury ,105. Calter CIRI ,107.Gentiana scabra Bunge., can inhibit and attenuate the expression of TLR4, MyD88 mRNA, and nuclear translocation of NF-κB in brain tissue, as well as the levels of IL-1β, TNF-α, and IL-6 in serum, suggesting that gentianine may reduce brain tissue injury due to ischemia/reperfusion by inhibiting TLR4 pathway-mediated inflammatory response [TLR4, also known as CD284, is a transmembrane protein in the Toll-like receptor family . During response . Procyanresponse .2+ generated during cerebral ischemia-reperfusion, thereby reducing brain cell apoptosis [Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey.) and Cardamonin  activates the HIF-1α/VEGF signaling pathway in rats with ischemia-reperfusion injury, and upregulate the protein expression of HIF-1α and VEGF, thereby increasing cerebral microvascular density and promoting intracerebral revascularization, and promoting angiogenesis, neural repair and functional recovery in MCAO rats [HIF-1α is a transcription factor that is widely distributed in mammals under hypoxic conditions and can activate a variety of hypoxia-response genes (HRGs) expression to regulate the oxygen homeostasis and energy metabolism balance of cells and organism . HIF-1α-poptosis , and canpoptosis . CatalpoCAO rats ,119.IS is a serious life-threatening disease associated with high rates of disability and mortality. Due to the rapid onset of the disease, there are many delayed factors in the treatment process, making early thrombolysis challenging to implement, thus affecting the outcome of neural health and functioning post-stroke. In recent years, a large amount of literature has shown that TCM can significantly improve IS with few side effects, demonstrating TCM’s potential benefits and significant potential for future development. TCM can regulate the signaling pathways to treat IS, such as JAK/STAT, NF-κB, MAPK, Notch, Nrf2, PI3K/Akt, which maintain BBB function, decrease brain edema, regulate energy metabolism, promote antioxidation, anti-inflammatory, and anti-apoptosis, reducing excitatory amino acid toxicity, enhancing neurogenesis, angiogenesis, and synaptogenesis.By summarizing and analyzing the natural compounds of traditional Chinese medicine that treat IS, we found that there are mainly flavonoids, alkaloids, polysaccharides, saponins, polyphenols, and terpenoids. The mechanism is mainly related to anti-oxidation, anti-inflammation, anti-apoptosis, and improving the permeability of BBB. The regulation effect of natural compounds from TCM on IS-related signaling pathways is shown in Despite the positive therapeutic effect of TCM on IS, there are still numerous challenges to overcome. Most of the research on TCM in IS models is still in the primary experimental stage, with few clinically applicable products. Furthermore, only known signaling pathways and related protein targets have been shown to play an important role in IS. However, there may be other pathway targets that have not yet been discovered. A comprehensive strategy combining metabolomics and network pharmacology is an effective tool to discover the targets and signaling pathways of TCM against cerebral ischemia-reperfusion. This strategy has been applied to TCM research, including drug target discovery, efficacy evaluation and mechanism research ,138,139.


Rhodoluna belongs to the ubiquitous freshwater bacterioplankton tribe Luna1-A2. Here, we report the complete sequences of Rhodoluna sp. strains KAS3 and KACHI23, which were isolated from freshwater lake and river surface water in Japan.The genus  Rhodoluna, which belongs to the Luna1-A2 tribe, have worldwide distribution, with sometimes relatively high abundance in the sequence databases (Rhodoluna lacicola and Rhodoluna limnophila) have been described  and KACHI23 (JCM 32204), which were isolated from a shallow eutrophic lake surface water sample collected from Lake Kasumigaura, Tsuchiura, Ibaraki, Japan , on 10 November 2010 and a river surface water sample collected from the Ichino River (Kachi Bridge), Yoshimi, Saitama, Japan , on 2 July 2013, respectively. The samples were filtered through a disposable syringe equipped with a 0.7-μm particle-retention glass-fiber filter . After filtration, the filtrates were spread on modified Reasoner’s 2A (MR2A) agar plates and incubated at 25°C for 2 to 5 days  and assembled using the assembler Canu v2.1.1 with specified parameters minReadLength=2200 and minOverlapLength=2200 (dnaA using DFAST (https://dfast.nig.ac.jp)  . Defaultppk), which is associated with intracellular accumulation of polyphosphate, was identified, in addition to a gene encoding a putative xanthorhodopsin-like protein associated with light-driven, proton-pumping rhodopsin. Furthermore, these strains also contained genes encoding putative alanine dehydrogenase-like proteins associated with ammonification.Based on annotation results for the KAS3 and KACHI23 genome sequences, a gene predicted to encode polyphosphate kinase (AP026910 and AP026911, which are linked to the BioProject accession no. PRJDB14318, BioSample accession no. SAMD00533725 and SAMD00533726, and DDBJ Sequence Read Archive accession no. DRR410665 and DRR410666, respectively. The details are summarized in The chromosome sequences and reads for strains KAS3 and KACHI23 were deposited in the GenBank/ENA/DDBJ database under the accession no.