Fibrosis accompanies most heart diseases and is associated with adverse patient outcomes. Transforming growth factor (TGF)β drives extracellular matrix remodelling and fibrosis in the failing heart. Some members of the ADAMTSL  family of secreted glycoproteins bind to matrix microfibrils, and although their function in the heart remains largely unknown, they are suggested to regulate TGFβ activity. The aims of this study were to determine ADAMTSL2 levels in failing hearts, and to elucidate the role of ADAMTSL2 in fibrosis using cultured human cardiac fibroblasts (CFBs). Cardiac ADAMTSL2 mRNA was robustly increased in human and experimental heart failure, and mainly expressed by fibroblasts. Over-expression and treatment with extracellular ADAMTSL2 in human CFBs led to reduced TGFβ production and signalling. Increased ADAMTSL2 attenuated myofibroblast differentiation, with reduced expression of the signature molecules α-smooth muscle actin and osteopontin. Finally, ADAMTSL2 mitigated the pro-fibrotic CFB phenotypes, proliferation, migration and contractility. In conclusion, the extracellular matrix-localized glycoprotein ADAMTSL2 was upregulated in fibrotic and failing hearts of patients and mice. We identified ADAMTSL2 as a negative regulator of TGFβ in human cardiac fibroblasts, inhibiting myofibroblast differentiation and pro-fibrotic properties. Cardiac fibrosis results from dysregulated deposition of extracellular matrix (ECM) molecules by activated cardiac fibroblasts (CFBs), termed myofibroblasts, leading to tissue stiffening7. Transforming growth factor (TGF)β is a central signalling molecule in cardiac development and disease, and a major driver of cardiac myofibroblast differentiation and fibrosis10. Inactive TGFβ, bound to latent TGFβ binding proteins (LTBPs), is stored in the ECM as the large latent complex (LLC), tethered to microfibrils12. Release from the inactive complex is required for TGFβ activation and initiation the pro-fibrotic response10. Thus, identification of molecular players governing TGFβ signalling in the heart is essential to develop novel treatment strategies that can counteract cardiac fibrosis.Fibrosis is a hallmark feature of heart failure, a leading cause of morbidity and mortality worldwide, and the degree of cardiac fibrosis is a strong predictor of poor outcomes in patients14. Some ADAMTS proteases have important roles in the heart17, but little is known about the role of ADAMTSLs. Emerging data suggests that they can bind and regulate fibrillin microfibrils20, major ECM components that control TGFβ bioavailability. Recessive loss-of-function ADAMTSL2 mutations cause geleophysic dysplasia (GD), an inherited connective tissue disorder resulting in severe musculoskeletal, pulmonary, and cardiac anomalies, with increased TGFβ levels and activity observed in patient-derived skin fibroblasts22. In beagles, an ADAMTSL2 founder mutation causes Musladin-Lueke Syndrome with severe skin and intermuscular fibrosis23. Adamtsl2−/− mice fail to survive past birth, likely as a result of lung anomalies associated with bronchial fibrillin microfibril accumulation, and have cardiac developmental defects24. Collectively, these data suggest that ADAMTSL2 regulates ECM deposition and TGFβ signalling and may thus have an important role in cardiac fibrosis and heart failure.Members of the ADAMTSL  family of secreted glycoproteins structurally resemble ADAMTS proteases, but lack enzymatic activity, as they do not have a protease domainIn the present study, we investigated expression of ADAMTSLs in heart failure, and identified novel functions of ADAMTSL2 in CFBs. Specifically, we found that ADAMTSL2 was robustly up-regulated in clinical and experimental heart failure, and ADAMTSL2 was predominantly expressed by CFBs. In cultured foetal and adult human CFBs, ADAMTSL2 negatively regulated TGFβ signalling, and attenuated myofibroblast differentiation and pro-fibrotic properties of CFBs.25, and in summary show concentric hypertrophic remodelling at two and four weeks post-AB, where the heart muscle is thickened, and end-stage dilated heart failure at 20 weeks, with thinning of the heart muscle, compared to sham-operated controls. Cardiac fibrosis (increased collagen expression and extracellular deposition) and increased TGFβ signalling was present at all three time-points, with increased α-smooth muscle actin (α-SMA) observed at two weeks25. We found that all members of the Adamtsl family were upregulated after AB, except Adamtsl6, which was unchanged. Adamtsl2 showed the highest up-regulation with a four–eightfold increase during hypertrophic remodelling, and an eightfold increase at end-stage cardiac dilatation  of mice subjected to AB for two, four and 20 weeks. The resulting cardiac phenotypes were previously reportedion Fig. a, suggesAdamtsl2 mRNA induction, showing a 2-3.5-fold increase in full-length protein at two, four and 20 weeks post-AB  after AB, compared to modest expression of Adamtsl2 in the sham-operated mice  in the LV wall , the endothelial cell marker von Willebrand Factor (Vwf), and the fibroblast markers type I Collagen (Col1a2), Periostin (Postn), and α-SMA (Acta2)  in the mouse heart  or vehicle control (Veh) in hfCFBs. Successful over-expression was confirmed by increased ADAMTSL2 mRNA   or at day four of a seven-day protocol (mature ECM)  and periostin (POSTN)28, were reduced 30–50% in L2 cells in both culture protocols  50–70% in the two protocols , recombinant, active TGFβ1 was added to Veh and L2 cells. Although L2 cells had lower levels of pSMAD at baseline, we found that TGFβ treatment increased pSMAD in both Veh and L2 cells, with a higher pSMAD increase in L2 cells relative to respective baseline levels , fibrillin-2 (FBN2) and tropoelastin (ELN) in L2 cells. The mRNA level of FBN1 was reduced to 80% of control in the immature ECM culture, and ELN to 40–60% of controls in both cultures, while mRNA levels of FBN2 were increased 1.2-fold in both cultures , contactin-1 (CNTN1), E-cadherin (CDH1) and tenascin C (TNC) were down-regulated, while adhesion molecules related to inflammation, vascular cell adhesion molecule-1 (VCAM1), L-selectin (SELL) and P-selectin (SELP), were among the most up-regulated. Collagen degrading enzymes such as matrix metalloproteinase 1 and 13 , as well as non-fibrillar collagens  were also among the up-regulated. Strikingly, osteopontin mRNA (SPP1) was down-regulated to 6% of controls , mini-chromosome maintenance protein 2 (MCM2) and marker of proliferation Ki-67 (KI67) were reduced to 50–80% of controls , which had demonstrably higher levels of ADAMTSL2 Fig. a, or conium Fig. b. Furtheium Fig. c–d. To dium Fig. e. Thus, ADAMTSL2 mRNA levels in myocardial biopsies from three cohorts of patients, namely aortic stenosis (AS), hypertrophic obstructive cardiomyopathy (HOCM) and dilated cardiomyopathy (DCM) vs. respective controls. Patient characteristics were reported previously, with hypertrophic remodelling, fibrosis and heart failure with preserved ejection fraction (HFpEF) in AS and HOCM, and dilated heart failure with reduced ejection fraction (HFrEF) and fibrosis in DCM patients32. Importantly, ADAMTSL2 mRNA was increased 2.5-fold in LVs of HOCM, DCM and AS patients . From the GTEx project of deceased organ donors, it was evident that ADAMTSL2 was expressed in human hearts, both in left ventricle and atrial appendage  in humans, a severe connective tissue disorder with poor prognosis, in which cardiac anomalies, such as progressive valve thickening, are found43. We speculate that elevated levels of ADAMTSL2 in patients and mice with fibrosis and heart failure could be beneficial, similar to BNP. The lack of experiments examining the in vivo role of ADAMTSL2 in cardiac remodelling, fibrosis and failure is therefore a significant limitation of our study. Such experiments are complicated by the fact that Adamtsl2−/− mice are non-viable24. Nevertheless, heterozygous Adamtsl2−/+ mice, or mice with global post-natal or fibroblast-specific conditional Adamtsl2 inactivation could be utilized. Efforts to increase ADAMTSL2 experimentally during heart failure progression, e.g. with an AAV vector, should also be made to evaluate the effects of ADAMTSL2 in vivo. Thus, whether increased cardiac ADAMTSL2 has beneficial effects in the diseased heart remains to be elucidated in future experiments.In the failing heart, adaptive brakes are expressed to counteract the maladaptive drivers. A prime example of an adaptive brake is the hallmark heart failure blood and tissue biomarker brain natriuretic peptide (BNP), which was recently included in the treatment guidelinesIn conclusion, the extracellular matrix-localized glycoprotein ADAMTSL2 was upregulated in fibrotic and failing hearts of patients and mice. ADAMTSL2 inhibited TGFβ and myofibroblast differentiation, reduced the expression of pro-fibrotic signature molecules and attenuated central pro-fibrotic properties of human CFBs. Mechanistically, we showed that ADAMTSL2 over-expressing cells produce less TGFβ, and speculate that ADAMTSL2 inhibits TGFβ deposition and activation, contributing to a negative TGFβ feedback loop. We suggest that ADAMTSL2 may have anti-fibrotic effects in the failing heart.An expanded Supplementary Methods section is available in the Supplementary Information.44.The human cardiac biopsy protocol was approved by the Regional Committee for Medical Research Ethics , the South-Eastern Regional Health Authority of Norway, and was in accordance with the Declaration of Helsinki. Informed consent was signed by all patients and the next of kin of heart donors. Mouse protocols were approved by the Norwegian National Animal Research Committee , and conformed to the NIH Guide for the Care and Use of Laboratory Animals  and the ARRIVE guidelines for reporting of animal research32. For detailed method description, see Supplementary Methods 3.1.Left ventricular (LV) tissue biopsies were obtained at Oslo University Hospital, Norway, from patients with heart disease of three aetiologies, Aortic stenosis , hypertrophic obstructive cardiomyopathy  and dilated cardiomyopathy . All patients received standard clinical evaluation, treatment and follow-up in accordance with Oslo University Hospital guidelines. Patient characteristics have been previously described25. In brief, experimental heart failure was induced in 8–10 week-old C57BL/6 J mice by aortic banding (AB) of the ascending aorta, causing pressure overload of the LV. Hearts were harvested at two, four and 20 weeks post-AB. Analgesia was administered pre- and post-operatively by subcutaneous injection of (0.3 mg/mL) buprenorphine, with additional analgesics given based on the status of the animal. The mice were euthanized by dissection of the heart under deep terminal anaesthesia breathing 3% isoflurane. For detailed method description, see Supplementary Methods 3.2.Mouse heart samples used for this study were derived from a previously published cohortAdamtsl2. Hematoxylin was used as the counterstain. For full method description, see Supplementary Methods 3.3.In situ hybridization was performed on sectioned, paraffin-embedded, AB- or sham-operated mouse hearts using an RNAscope technology with a probe specific for mouse 45. Briefly, CMs and CFBs were isolated from hearts of 1–3 day old neonatal rats (Wistar). Cells were plated at 3.8 × 104 cells/cm2cultured in serum-containing Dulbecco’s Modified Eagle medium in a 37 °C, 5% CO2 humidified incubator. The purity of the cultures was determined by expression analysis of the CM-specific gene Tnni3, the endothelial cell marker Vwf, and the fibroblast markers Col1a2, Postn, and Acta2  or 10,000 cells/cm2 and cultured for seven days (generating a mature ECM) before harvest  or vehicle control (Veh) was performed one or four days after seeding in cultures with immature or mature ECM, respectively. Transduction was performed in serum-containing medium for 24 h, followed by serum-free medium for 48 h. Non-transduced cells were treated with conditioned medium, containing ADAMTSL2 protein (L2-medium), or control (Veh-medium) diluted 1:1 in serum-free medium, for three days. Cells were harvested 72 h after seeding. Transduced cells were treated with recombinant TGFβ (10 µg/µL) for 24 h following 24 h serum starvation. Non-transduced cells were treated with recombinant TGFβ (10 µg/µL) and/or TGFβ-SMAD inhibitor (10 µM) for 2 h following 24 h serum starvation. Non-transduced cells were treated with L2 or Veh conditioned medium, pre-incubated for 1 h with recombinant TGFβ1 (10 µg/µL), for 0, 30 and 60 min before harvest. For detailed method description, see Supplementary Methods 3.5.Commercially available human foetal (Cell Applications) and adult (PromoCell) cardiac fibroblasts  were used for cell culture experiments with the recommended culture media. In summary, cells were plated at 20,000 cells/cmhfCFBs were cultured on coverslips, fixed in 4% PFA, permeabilized and incubated with primary antibodies for α-SMA, fibrillin-1, EDA-fibronectin, LTBP1 and Collagen type I, and fluorescently labelled secondary antibodies. Cells were mounted onto slides and proteins were visualized with the Axioscan Z1 (Carl Zeiss) for full slide scanning, or the LSM 710 confocal microscope (Zeiss). For full method description, see Supplementary Methods 3.6.hfCFBs transduced with Veh or L2 were mixed with collagen and added to BSA-coated plates. The gels were allowed to polymerize before serum-free medium was added to release the gels from the surface. Contraction was observed over the next 24 h, percent contraction was calculated by measuring the circumference of the collagen gels. For full method description, see Supplementary Methods 3.7.3 cells/cm2 in 96-well plates. After 24 h, serum-free medium, or serum-containing medium for positive control, was added. After 48 h, cells were labelled with EdU (10 µM) for 2 h. Cells were fixed according to protocol  and fluorescence was measured on the Hidex microplate reader.hfCFBs transduced with Veh or L2 were seeded in serum-containing medium at 10hfCFBs were seeded and transduced in 12-well plates, and kept in serum-free medium for 24 h. A 1 mm wide scratch was made through the cell monolayer using a 200 µL pipette tip and fresh serum-free medium was added. Cell migration across the gap was observed and images were taken with the Eclipse Ts100 phase contrast microscope. Percent migration was calculated from the size of the scratch at 24 h using ImageJ (NIH).3H]-Proline  was added. After 48 h, cells were lysed in NaOH (1 M) and diluted in OptiPhase HiSafe 3 liquid scintillation cocktail . Amount of incorporated radiolabelled proline, a surrogate for collagen biosynthesis46, was measured on the Wallac Winspectral 1414 liquid scintillation counter (Perkin Elmer).hfCFBs were seeded and transduced in 12-well plates. After 24 h, serum-free medium containing ascorbic acid (50 µM/mL) and L-[2,3-RPL32), L4 (RPL4), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). For full method description, see Supplementary Methods 3.8.Total RNA was isolated from LVs and cell cultures, and relative gene expression was determined using TaqMan probes  was used to mine for published single cell RNA sequencing data describing Adamtsl2 expression in different organs and cell types. The Genotype-Tissue Expression (GTEx) Project Portal  was used to analyse ADAMTSL family expression (RNA sequencing) in cultured dermal fibroblasts, atrial appendage and left ventricle of deceased organ donors. Charts were created using the interactive graphics functionality of the databases.The Single Cell Expression Atlas database  according to the manufacturer’s protocol. Secreted proteins were harvested from the cell culture medium. Supernatants and lysates were stored at − 20 °C. N-linked oligosaccharides were enzymatically removed from glycosylated proteins using PNGaseF. Western blotting was performed using the Trans-Blot Turbo blotting system (Bio-Rad). Membranes were blocked in 5% non-fat dry milk, casein or BSA, before incubation with primary antibodies (see Supplementary Table Protein lysates from mouse and human LVs were extracted as previously describedt-test when comparing two groups, the one-way ANOVA with Tukey’s multiple comparisons test when comparing multiple groups, and the two-way repeated measures ANOVA with the Geisser-Greenhouse correction when comparing multiple time-points. P-values < 0.05 were considered statistically significant, and exact p-values are given in the figures, unless P < 0.001.Data are expressed as the minimum value, group mean, and maximum value, with all data points shown in graphs, relative to respective controls and to a reference gene/protein. Normal data distribution was evaluated using the Shapiro–Wilk test. Statistical differences were tested in GraphPad Prism 8, using the unpaired Student Supplementary Information.


Dexmedetomidine has sympatholytic, anti-inflammatory, and analgesic effects and may exert anti-tumor effect by acting on α2A adrenoreceptor. We investigated whether perioperative dexmedetomidine preserves immune function in patients undergoing uterine cancer surgery.One hundred patients were randomly assigned to the control or dexmedetomidine groups (50 patients each). Dexmedetomidine was infused at rates of 0.4 μg/kg/h intraoperatively and 0.15 μg/kg/h during the first 24 h postoperatively. The primary outcome was natural killer (NK) cell activity, which was measured preoperatively and 1, 3, and 5 days postoperatively. The inflammatory response was measured by interleukin-6, interferon-γ, and neutrophil/lymphocyte ratio, and pain scores and opioid consumption were assessed. Cancer recurrence or metastasis and death were evaluated 2 years postoperatively.P=0.496). Interferon-γ increased postoperatively in the dexmedetomidine group, whereas it maintained at the baseline value in the control group. Change in interferon-γ differed significantly between groups (P=0.003). Changes in interleukin-6 and neutrophil-lymphocyte ratio were comparable between groups. Both pain score with activity during the first 1 h and opioid consumption during the first 1–24 h postoperatively were lower in the dexmedetomidine group. Rates of cancer recurrence/metastasis  and death within 2 years postoperatively  were not different between groups.NK cell activity decreased postoperatively in both groups and changes over time were not different between groups (Perioperative dexmedetomidine had no favorable impacts on NK cell activity, inflammatory responses, or prognosis, whereas it increased interferon-γ and reduced early postoperative pain severity and opioid consumption in uterine cancer surgery patients. Although surgical resection is the main and curative treatment for solid tumors, the spread of tumor cells in the blood and lymphatic system might occur by surgical manipulation . SurgicaDexmedetomidine is a highly selective α2 adrenoreceptor agonist and has broad pharmacologic effects including anesthesia, analgesia, sedation, and anxiolysis . PeriopeGynecologic cancer contributes significantly to the morbidity and mortality of females worldwide , and cerThis study was approved by the Institutional Review Board and Hospital Research Ethics Committee of Severance Hospital, Yonsei University Health System, Seoul, Korea (#4-2015-0453) and registered at ClinicalTrials.gov (NCT02896413). Inclusion criteria were women 20–65 years old, who had American Society of Anesthesiologists (ASA) physical status classification of I–III and underwent elective surgery for uterine cancer. Exclusion criteria were renal or hepatic impairment, immunosuppressive therapy, immune system disorders, or cancer metastasis. Informed consent was obtained from all patients before participating in this study.In total, 100 patients were enrolled and randomly assigned into one of the study groups (50 patients each) using a computer-generated random numbers table. In the dexmedetomidine group (DEX group), dexmedetomidine was infused at 0.4 μg/kg/h from anesthetic induction to the end of surgery and continued at 0.15 μg/kg/h for the first 24 h postoperatively. The dose of dexmedetomidine was determined based on that of previous studies showing no hemodynamic instability or deep sedation . In the After monitors including electrocardiography, pulse oximetry and blood pressure monitor were applied, anesthesia was induced with propofol 1–2 m/kg and remifentanil 1–2 μg/kg. Rocuronium 0.6 mg/kg was administered to facilitate endotracheal intubation. Anesthesia was maintained with 4%–7% desflurane and remifentanil 0.05–0.1 μg/kg/min to maintain the mean arterial pressure within 20% of the preoperative value and the bispectral index between 40 and 60. Body temperature was maintained at 36.5 ± 0.5°C throughout surgery. At 15 min before the end of surgery, all patients received fentanyl 50 µg and ramosetron 0.3 mg for prevention of postoperative pain and nausea/vomiting. At the end of surgery, patients received neostigmine 1 mg and glycopyrrolate 0.2 mg for reversal of residual neuromuscular blockade. For postoperative analgesia, all patients received intravenous patient-controlled analgesia (IV-PCA) consisting of fentanyl 15 μg/kg and ramosetron 0.3 mg . Intravenous ketorolac 30 mg was administered three times per day on the day of surgery. Additional analgesics were available for patients having an 11-point numeric pain rating scale score ≥4 or requesting supplemental analgesics: intravenous fentanyl 50 μg or pethidine 25 mg in the post-anesthesia care unit and pethidine 25 mg or tramadol 50 mg in the postoperative ward. Drugs possessing anti-inflammatory effects  were not administered during the first 48 h postoperatively. An investigator unaware of the group assignment evaluated possible dexmedetomidine-related adverse effects .The primary outcome measure was NK cell activity, which was measured preoperatively and on postoperative days (PODs) 1, 3, and 5. NK cell activity was analysed using the NK Vue kit . One mL of whole blood was drawn into a NK Vue tube containing Promoca (a cytokine that stimulates NK cell activity) and RPMI 1640 media and then incubated at 37°C for 24 h. This selected stimulatory cytokine and incubation period allows NK cells to secrete interferon-γ (IFN-γ) preferentially over other immune cells, and the supernatant IFN-γ level measured by NK Vue ELISA may be an indicator of NK cell activity. We calculated the mean IFN-γ value from duplicate readings.Other outcome measures included inflammatory responses assessed by interleukin-6 (IL-6), IFN-γ, and neutrophil-lymphocyte-ratio (NLR), which were measured preoperatively and on POD 1, 3, and 5. Pain severity and opioid requirement were assessed 1, 24, and 48 h postoperatively. Pain severity was evaluated using an 11-point numerical scale . The opioid requirement was assessed by IV-PCA fentanyl dose and additional opioid consumption . Rates of cancer recurrence or metastasis and death were assessed 2 years after surgery.The sample size was calculated based on a previous study showing a reduction of NK cell activity on POD 1 (compared with baseline) of 83.1 ± 25.2% . Forty-et-test or Mann-Whitney U test after testing for normality of distribution using the Kolmogorov-Smirnov test and expressed as mean ± SD or median (interquartile range). Categorical variables were analysed using χ2 or Fisher exact tests and expressed as absolute number (percentage). Variables measured repeatedly, such as NK cell activity, INF-γ, IL-6, and NLR, were analysed using a linear mixed model, with patient indicator as the random effect and group, time, and group-by-time as the fixed effects, after log-transformed for normality of distribution. Post-hoc analyses with Bonferroni correction were performed when variables measured repeatedly showed significant differences between groups. A P value <0.05 was considered statistically significant. Statistical analyses were performed with the Statistical Package for Social Sciences .Continuous variables were analysed using the independent Of 100 patients enrolled, 9 patients were eliminated. One patient in the Control group withdrew consent for participation and 4 patients did not meet the study protocol (they were anesthetized with propofol or sevoflurane instead of desflurane). The remaining 91 patients completed the study without any complications Table 1.P=0.113) and it decreased significantly below baseline after surgery in both groups. Linear mixed model analysis showed that the perioperative change of NK cell activity over time was not different between groups (P=0.697) (NK cell activity before surgery was comparable between groups (P=0.697) Figure 2P=0.777). Compared to the baseline, IFN-γ level increased after surgery and was higher on PODs 3 and 5 in the DEX group, whereas it was maintained in the Control group. The change of IFN-γ over time was statistically significant between groups (P=0.010). IFN-γ level on POD 3 was higher in the DEX group compared to the Control group.IFN-γ level before surgery was comparable between groups (P=0.002). In both groups, IL-6 increased after surgery, peaking on POD 1. Compared to the baseline, IL-6 level was higher on PODs 1, 3, and 5 in the DEX group and on POD 1 in the Control group. The change of IL-6 over time was not significant between groups (P=0.117).IL-6 level before surgery was lower in the DEX group than in the Control group (P=0.494) (NLR before surgery was similar between groups. It increased after surgery and was higher than baseline on PODs 1 and 3 in both groups. The change of NLR was not different between groups (P=0.494) Figure 3P=0.016). At other times, pain scores were not different between groups. Fentanyl IV-PCA dosage during the first 48 h postoperatively was comparable between groups. Additional opioid consumption 1–24 h postoperatively was lower in the DEX group than in the Control group . At other times, additional opioid consumption  was similar between groups (Pain score during activity (sitting up) 1 h postoperatively was lower in the DEX group than in the Control group . Death occurred in 1 patient (2.2%) in the DEX group (due to cancer recurrence with lung metastasis) and 3 patients (6.7%) in the Control group (due to cancer recurrence in 1 patient and cancer recurrence with lung metastasis in 2 patients) (Four patients (8.7%) in DEX group and 7 patients (16.3%) in the Control group had cancer recurrence and/or metastasis during the 2-year follow-up period (atients) Table 3.Dexmedetomidine administration in patients undergoing uterine cancer surgery did not demonstrate a favorable impact on immunity in terms of perioperative changes of NK cell activity, IL-6, and NLR. However, dexmedetomidine was associated with higher IFN-γ postoperatively and reduced both pain severity and opioid requirement early postoperatively. Although statistically insignificant, rates of cancer recurrence/metastasis (8.7 vs. 16.3%) and death (2.2 vs. 6.7%) within 2 years after surgery were much lower in the dexmedetomidine group.Major oncologic surgeries with extensive resection impair immunity, by causing sympathetic hyperactivation and excessive inflammation . It is iPerioperative immune dysfunction includes profound suppression of cell-mediated immunity, expressed as a decrease in the number and activity of immunocompetent cells such as NK and T cells . NK cellExaggerated inflammatory responses with excessive production of proinflammatory cytokines induced by surgical trauma also contribute to immune dysfunction . DexmedeInterestingly, dexmedetomidine was associated with a higher IFN-γ level postoperatively. IFN-γ is produced by activated T cells and NK cells in response to immune stimuli and enhances cellular immune immunity . It exervia the hypothalamic-pituitary-adrenal axis  and registered at ClinicalTrials.gov (NCT02896413). The patients/participants provided their written informed consent to participate in this study.JC: Conceptualization, Funding acquisition, Investigation, Data curation, Formal analysis, Writing- original draft. KS: Investigation, Data curation, Formal analysis. M-YK: Investigation, Data curation, Formal analysis. SK and YY: Conceptualization, Supervision, Writing - review & editing. JC, MK, SK, and YY: Agreement to be accountable for all aspects of the work. All authors contributed to the article and approved the submitted version.This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2016-0107).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from  Escherichia coli, CRP  regulates the transcription of about 400 genes  + [P] + DK) − (([L] + [P] + DK)2 − 4·[L]·[P])0.5)/2·[P], where ΔF, ΔFM, [P], and [L] represent fluorescence change, the maximal fluorescence change, total protein concentration, and total ligand concentration, respectively [cor = F × 10(P + ΔA)/2, where F and Fcor denote fluorescence intensity before and after the correction, whereas P and ΔA indicate the initial sample absorption at the excitation wavelength and the change in absorption introduced by ligands, respectively [Fluorescence intensities were measured at 25 °C using a F-4500 Fluorescence Spectrophotometer (Hitachi corporation). H-NS and CRP in 50 mM MOPS buffer, pH 7.5 were excited at 280 nm and the emission spectra were recorded from 290 to 400 nm under various ligand concentrations. The dissociation constant, ectively ,35. The ectively .E. coli CRP, occurring as cAMP- or cGMP-bound forms, were used for docking simulation. The docking models were prepared by removing the corresponding ligands from the structures. The prepared ppGpp (CID: 135398636) and the CRP models were simulated for the protein-ligand docking. The docking, energy minimization, and scoring of the results were performed using GalaxyWEB server as described previously [The two structures (1g6n and 1n9i) of eviously ,38,39 Theviously ,41.


Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets.The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature’s toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell’s ability to cope with environmental stress.  Candida albicans causes ~750,000 cases of invasive life-threatening bloodstream infections in immunocompromised patients worldwide each year , a ubiquitous and highly conserved regulator of the cellular protein homeostasis. Hsp90 regulates cell morphology, drug resistance, and virulence in albicans  and otheoformans , which cumigatus  which tan rubrum , and Spochenckii , the cauic cells  and biofic cells , switchiic cells , inductiic cells , and delic cells . Althougtoxicity . Targetitoxicity . Speciesg pocket , could pSaccharomyces cerevisiae  play a crucial role in regulating Hsp90. Their impact on chaperone activity, directionality of the chaperone cycle, and binding to co-chaperones has been extensively studied in mammalian cells and the model eukaryote revisiae . Howeveralbicans , as wellumigatus .S. cerevisiae Hsp90 negatively affects the chaperone cycle, attenuates interactions with clients and co-chaperones, induces conformational switching, and increases sensitivity to Hsp90 inhibitors , which has 65% sequence identity with the S. cerevisiae homolog, results in resistance to the broadly deployed antifungal fluconazole  subunit of CK2  and the corresponding wild type (YSD673), strains were grown to mid-log phase at 30°C in 200 ml volumes in YPD at 30°C. Whole cell proteins were extracted as described below and the C-terminally TAP-tagged chaperone was pulled down using IgG agarose beads from lysate containing 20 mg of protein (see below). The protein was eluted in 100 µl 1x sample buffer and heated to 95°C for 10 minutes prior to loading 30 µl onto a 10% Tris-glycine SDS-PAGE gel for separation. Coomassie-stained gels were imaged and the bands corresponding to Hsp90 were excised and processed by in-gel digestion . Hsp90’sC. albicans Hsp90 monomer was generated based on the S. cerevisiae Hsp90-Sba1 crystal structure . 50 ml of cell culture were pelleted, snap frozen in liquid nitrogen, and stored overnight at -20°C. Pellets were thawed on ice, washed twice with 1x PBS and resuspended in 250 µl lysis buffer . Cells were transferred to Precellys 2 ml Tough Micro-organism Lysing Kit tubes (VK05) and lysed using a Precellys Evolution tissue homogenizer. Samples were processed in 3 cycles, each consisting of bead beating at 6,000 rpm for 1 minute, pause for 30 seconds, bead beating at 6,000 rpm for 1 minute. Tubes were rested on ice for at least 1 minute between each cycle. Lysates were cleared twice by centrifugation at 20,000 xg at 4°C for 30 minutes, and 5 minutes. Supernatants were transferred to microfuge tubes and refrigerated in preparation for sample preparation and gel loading. The lysates’ whole cell protein concentrations were determined with a Quick Start Bradford Protein Assay (Bio-Rad) and calibrated against a BSA standard curve. Samples were diluted to 0.1 µg/µl protein in sample buffer , heated at 95°C for 10 minutes and either cooled on ice prior to loading or stored at -20°C.To determine Hsp90 stability in the wild-type, As loading controls, 100 µl per sample were loaded onto a 10% SDS-PAGE gel and separated for 20 minutes at 90 volts, followed by 100 minutes at 120 volts. To visualize proteins, gels were stained with SimplyBlue SafeStain (Invitrogen) according to the manufacturer’s instructions and imaged by scanning.C. albicans Hsp90, 15 µl were loaded on a 10% SDS-PAGE gel and separated for 20 minutes at 90 volts, followed by 100 minutes at 120 volts. Proteins were wet-transferred onto a PVDF membrane (Bio-Rad) for 135 minutes at 300 mA in a Mini-PROTEAN Tetra Vertical Electrophoresis Cell filled with transfer-buffer . To block free sites, membranes were treated with PBST  with 0.1% non-fat milk overnight at 4°C. The blots were probed for 1 hour at room temperature with α-CaHsp90 . Cells were transferred to 15 ml Precellys sample tubes (VWR BERT KT03961-1-4061), mixed with 1 ml acid-washed glass beads (~400 µm) and mechanically disrupted using a Precellys Tissue Homogeniser. Tubes were agitated three times for one minute with one minute on ice in between. The lysate was cleared by centrifugation at 20,000 xg twice for 5 minutes at 4°C and protein concentrations were determined using the Bradford assay described above. 7.5 mg of protein were mixed with 100 µl of IgG or α-HA agarose, depending on the strain’s genotype, in co-IP washing buffer  and gently rotated for 2 hours at 4°C. Following precipitation, the agarose beads were washed carefully 3-5 times with 1 ml co-IP washing buffer before being mixed with 100 µl 1x sample buffer  SDS, 36% glycerol, 5% 2-mercaptoethanol and 0.012% bromophenol blue). Prior to loading, samples were heated at 95°C for 10 minutes and cooled on ice ready for loading or stored at -80°C. Proteins were separated at 120 volts on size-appropriate SDS-PAGE gels.To purify Hsp90 for mass-spectrometric analysis and to investigate the effect of Hsp90 phospho-mutant alleles on co-chaperone binding, 200 ml of cells were grown as described above, washed twice in 1x PBS and resuspended in 2 ml co-IP lysis buffer , 0.25% deoxycholate Na, 150 mM NaCl, 5 mM EDTA, 1 mM NaTo visualize epitope-tagged co-chaperones, proteins were wet-transferred onto PVDF membranes for 16 hours at 20 volts in a Mini-PROTEAN Tetra Vertical Electrophoresis Cell filled with transfer-buffer . Membranes were blocked with PBST and 5% milk for at least 1 hour at room temperature before being probed with primary and secondary antibodies. To visualize TAP-tagged Sti1, membranes were treated with α-TAP antibody (Open Biosystems #CAB1001). Membranes carrying HA-tagged Cdc37, Aha1 or Sba1 were exposed to α-HA antibody (Invitrogen #71-5500). Both primary antibodies were diluted 1:5,000 in PBST with 0.2% milk and subsequently conjugated with α-rabbit antibody-HRP conjugate (BioRad Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate #1705046) diluted 1:5,000. Membranes were incubated in antibody solutions for 15 minutes each and washed at least three times with 1x PBST using the SNAP i.d. 2.0 Protein Detection System. Lastly, proteins were visualized with Clarity Western ECL (BioRad).To assess susceptibility to thermal stress, optical densities of stationary phase cultures were measured at 600 nm and adjusted to 0.1 in 1x PBS and serially diluted two-fold. 4 µl of cell suspension were then spotted onto solid YPD or YPM and plates were incubated at the indicated temperature for 48 hours. Plates were photographed and analyzed visually.Stationary phase cells grown in YPM at 30°C were diluted to an optical density of 0.2 in YPD or YPM and grown for 16 hours at 30°C while shaking at 200 rpm. In preparation for microscopy, cells were washed and diluted with 1x PBS to a cell density appropriate for microscopy. Images were collected using the DIC settings on a Nikon Eclipse E8000 microscope with a Nikon Digital Sight DS-5M camera. To calculate the morphology index (MI) , a total3 cells/100 µl. Fluconazole was diluted in water to a starting concentration of 512 µg/ml and 20 µl of drug were mixed with 180 µl YPD or YPM in a 96-well flat bottom dish. The drug was then diluted two-fold in YPD or YPM to cover a gradient ranging from 256 µg/ml to 4 µg/ml. 100 µl of cell suspension were added to each well and plates were incubated for 48 hours at 25°C and optical densities recorded at 595 nm. A 5 mM radicicol stock solution (DMSO) was diluted to 50 µg/ml in YPM or YPD. The drug was diluted in either media to the following concentrations: 1, 2, 3, 4, 5, 7.5, 10, 15, 20, and 25 µg/ml and strains inoculated as above. To account for the effects of DMSO alone, strains were also inoculated in 0.0275% DMSO. Plates were incubated for 48 hours at 25°C in the dark and optical densities recorded at 595 nm. Optical density values were normalized to the no treatment column and visualized using Java TreeView version 1.1.6r4  and incubated at 37°C for 24 hours either in flat-bottom 96-well dishes to quantify metabolic activity or on Thermanox™ coverslips to assess structural integrity via scanning electron microscopy. Metabolic activity, serving as a proxy for cell viability, was assessed by metabolic reduction of resazurin sodium salt (Sigma-Aldrich/Merck #R7017)  . For the #R7017) . To visu #R7017) .All analyses were carried out in R Version 3.6.0 and plotted using ggplot2. To assess the effect of the Hsp90 mutant alleles on cellular morphology, the MIs for samples grown in YPD and YPM were compared with each other using the Mann-Whitney U test. Statistical differences between biofilm metabolic activity were assessed by a linear model of strain versus optical density using Ordinary Least Squares regression. P-values were calculated from F statistics comparing the mutant strains to the maltose control.C. albicans Hsp90 in situ, the wild-type strain and four mutants, each lacking one CK2 subunit, were grown in rich media at 30°C upon which epitope tagged Hsp90 was purified and analyzed by mass spectrometry. Without further enrichment, Hsp90 sequence coverage in trypsin digests was 70% or higher and ranged between 40-45% in endoproteinase Glu-C digests. Three serine phosphorylation sites were identified using mass spectrometry , we decided to focus on this residue. Lack of phosphorylation of S530 in the cka2Δ/Δ mutant suggests this residue could be relevant for the increased fluconazole resistance previously observed in the cka2 mutant , species associated with trees (Neolecta irregularis), fermentative yeast (Nadsonia fulvescens), methylotrophic yeasts , halo-tolerant species  and the opportunistic pathogens . No serine alleles were detected amongst the 82 Agaromycotina, which include the human pathogen Cryptococcus neoformans as well as mushrooms and jelly fungi. In summary, the serine allele is rare; it is present in less than 5% of fungi. Serine occurs in species that are capable of causing disease in plants, thrive in extreme environments, and exist as commensals of the human body where they can cause opportunistic infections in responses to changes in the host’s immune status, like C. albicans.Given the divergent nature of the Hsp90 region S530 resides in  or a non-phosphorylatable alanine (A) while the remaining HSP90 wild-type allele was placed under the control of the maltose-inducible promoter MAL2p. Consequently, strains grown in dextrose solely expressed the mutant allele, thereby revealing the effects of phosphorylation. Growth in maltose resulted in expression of the wild-type allele, serving as control and for strain maintenance. Non-phosphorylatable and phosphomimetic alleles were created for both residues  and compared to the wild type and the MAL2-HSP90 promoter control, that carries two HSP90 wild-type alleles, one of which is under the control of the MAL2 promoter.To investigate the effects of phosphorylation of T25 and S530 and to accommodate the ‘obligate diploid’ nature of C. albicans Hsp90 C-terminus   media alone and supplemented with Calcofluor White or Congo Red . While deviating from our observation of a growth defect in the T25 mutants at 37°C on YPD agar, Azadmanesh et al. have shown that the physical environment influences C. albicans gene expression and development (Morphogenetic diversity is a key prerequisite for the formation of al drugs . To deteelopment .C. albicans cellular morphogenesis, we demonstrated that phosphorylation of S530 results in changes in colony morphology and reduced biofilm cell viability due to loss of Hsp90 function. Any manipulation of the T25 residue, however, leads to elongated cells, aberrant colony morphology and increased biofilm cell viability.Building on our finding that Hsp90’s phosphorylation status determines its effect on in vitro. Taken together, our results suggest that S530 is a novel phospho-switch. When phosphorylated, it blocks Hsp90 function and thus the expression of virulence traits, which may aid its adaptation to a commensal lifestyle while maintaining the ability to cause invasive infections. Alterations of T25, however, are important beyond phosphorylation, suggesting this residue to be a ‘switch point’, important for structural integrity of C. albicans Hsp90.In summary, Hsp90’s phosphorylation status affects fungal virulence traits S530 blocks expression of virulence traits while any alteration of Hsp90T25 resulted in a loss-of-function phenotype. This suggests that S530 is a novel C. albicans Hsp90 phospho-switch while T25 could act as switch point.Our data indicate that phosphorylation of Hsp90C. albicans wild type or CK2 mutants, we observed that any modification of T25, be it a non-phosphorylatable alanine allele or a phosphomimetic glutamic acid residue, negatively affects co-chaperone binding, survival of high temperatures, and susceptibility to drug and Hsp90 inhibition, which is similar to what has been described in S. cerevisiae and mammalian cells, (C. albicans Hsp90, although not necessarily a CK2 phosphorylation site. Switch points impact on the overall conformation and function of a protein through conformational disturbances (S. cerevisiae and T25 in C. albicans, as changes in the acetylation status of the neighboring conserved C. albicans K30 (S. cerevisiae K27 (Although we could not detect phosphorylation of T25 in the n cells, . Modelinurbances . These turbances . The effcans K30  or S. cesiae K27  residuesS530 in C. albicans hyphal cells (MAL2-HSP90 control are phenotypically more similar to the non-phosphorylatable mutant S530A than they are to the phosphomimetic S530E mutant (cka2Δ/Δ mutant encoding the α’ subunit of CK2, suggests that two of the active CK2 forms, α’ and α’2ββ’, are severely compromised and unable to target Hsp90. Alternatively, the blighted CK2 complex could fail to activate another kinase, which is responsible for phosphorylation of S530.Detection of Hsp90 phosphorylation sites varies greatly between different phospho-proteomic studies. Previous investigations identified between zero , nine C and 18 sal cells . This fiS. cerevisiae (C. albicans, it is conceivable that threonine could also be phosphorylated, suggesting mechanistic conservation. To date, only one record characterizing a homologous threonine residue exists. The S. cerevisiae homologous residue T533 was investigated using phosphomimetic and non-phosphorylatable alleles (C. albicans virulence traits in a commensal turned pathogen yeast, and the lack of involvement in Hsp90 function of T533 in the benign yeast, S. cerevisiae would be in keeping with S530 to be a genetic factor that reduces severity as predicted by the trade-off hypothesis of optimal virulence theory.Although S530 resides in a sequence divergent region of Hsp90 (revisiae , a close alleles . T533, hIn conclusion, Hsp90’s phosphorylation status and consequently structural integrity affect fungal virulence traits. CK2-mediated phosphorylation of the divergent S530 residue resulting in changes in fungal virulence traits could be further exploited as a novel therapeutic target to fight fungal infections.https://github.com/hobrien/DiezmannLab/blob/master/T25_analysis.Rmd.The datasets presented in this study can be found in the github online repository: in vivo assays. HOB analyzed growth, morphology, and biofilm data. CS assisted with mass spectrometric analyses. MB and GR performed and analyzed biofilm formation. JS modeled Hsp90 surfaces charges. SD drafted and edited the manuscript. All authors contributed to the article and approved the submitted version.LA, JLC, and SD conceived and designed this study. LA constructed strains, prepared samples for mass spectrometry, conducted Hsp90 stability assays and assessed co-chaperone associations. JLC conducted Hsp90 stability and drug susceptibility assays. UO performed the structural modeling analysis. RW performed Hsp90 phylogenetic analyses and ancestral character state reconstruction. CW performed This work was funded by MRC grant MR/L018349/1 to SD. JC was supported by the MRC GW4 ‘Biomed’ Doctoral Training Program.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear.We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis.O-mono-glucuronyl 18β-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 → 2)-glucuronyl] 18β-glycyrrhetinic acid increased AFP+ cell counts and albumin+ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect.GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-+ cell count and decreased Hes5+ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation.We evaluated the effects of (±)-2- propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased β-cateninIn conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling. •+ and albumin+ cell counts.Glycyrrhizin (GL) derivatives increased AFP•Glycyrrhizin promotes hepatic differentiation at the hepatoblast formation stage.•Mono glucuronide showed the most significant hepatic differentiation effect.•Glycyrrhizin promotes hepatic differentiation via T1R3 signaling.•Wnt and Notch signaling are involved in GL-induced hepatic differentiation. There are many types of liver diseases, e.g., viral hepatitis, alcoholic and nonalcoholic liver diseases, cholangiopathies, autoimmune liver disease, which may lead to advanced liver disease stages, cirrhosis or hepatic malignancies. Liver disease and cirrhosis are significant health disorders with limited therapeutic strategies available for their management. Aberrant extracellular release of high-mobility group box1 (HMGB1) is associated with many forms of liver disease  βGA (Glc) was isolated from the roots of Glycyrrhiza uralensis strain 83-555, according to a method previously described by Cokey Co [O-[apiosyl (1 → 2)-glucuronyl] βGA (Api) and licorice saponin H were isolated from commercially available licorice extracts using a method similar to that used for the extraction of Glc ) in a 5% COi et al. . The med4 cells/cm2 onto a Matrigel (GFR) -coated dish and cultured in an hES medium which was changed daily. To induce the differentiation of the definitive endoderm, 7 days after culturing (on day 0), the medium was replaced with an RPMI medium  supplemented with 100 ng/mL activin A  and changed every other day. To induce hepatic specification, the medium was replaced with an RPMI medium supplemented with 10 ng/mL bFGF and 20 ng/mL bone morphogenetic protein 4  on day 5 and changed every other day. Then, for hepatoblast formation, the medium was replaced with an RPMI medium supplemented with 20 ng/mL hepatocyte growth factor  on day 10 and changed every other day. GL and its derivatives were added on days 10–15, except otherwise mentioned.We induced hepatic differentiation, according to the method described by Si-Tayeb et al.  with min2.3+ cells, were represented as the relative value compared to that obtained following solvent control treatment.Cultured cells were dissociated using dispase (Thermo Fisher Scientific) and a cell dissociation buffer (Thermo Fisher Scientific). Cells fixed with Cytofix Fixation Buffer  were washed with PhosFlow Perm/Wash Buffer I (BD) and incubated with primary antibodies  at 4 °C overnight. After the cells were washed, they were incubated with Alexa 488 - and phycoerythrin -conjugated anti-rabbit and anti-mouse IgG, respectively at 4 °C overnight. Then, the cells were washed again and passed through a 45-μm filter, and were subjected to flow cytometric analysis using a FACS Calibur (BD) flow cytometer according to the manufacturer's instructions. For flow cytometric analysis, the proportion of immunopositive cells was determined using the antibodies mentioned above. The data, which were normalized to the population of HNF4α2.4t-test with a significance level of 0.05, and Fisher's protected least significant difference (LSD) test with a significance level of 0.025, were performed.Several statistical tests such as ANOVA followed by Tukey's post-hoc test, Student's 33.1+ cell count and slightly increased albumin+ cell count (data not shown). In addition, to determine the sensitive period of the cells to GL, the duration of treatment was modified as follows: (1) throughout the hepatic differentiation, for 15 days, or (2) only for the hepatoblast formation stage, i.e., the last 5 days of hepatic differentiation. AFP+ cell count significantly increased by GL treatment . Another aglycon, αGA, had no effect on hepatic differentiation. CBX, 3-O-hemisuccinyl βGA, also had no effect on hepatic differentiation. The glucuronate conjugates, GL, Mono, and Glc showed hepatic differentiation activity in a dose-dependent manner, and significantly increased AFP+ cell count at a concentration of 500 nM . Api tended to increase AFP+ cell count at a concentration of 5 and 500 nM. However, LSG and LSH had no effects on hepatic differentiation. No aglycone nor CBX had an effect on albumin+ cell count. In contrast, GL increased albumin+ cell count in a dose-dependent manner. Moreover, Glc and Mono significantly increased albumin+ cell count at a concentration of 500 nM  and 5 nM . Furthermore, GL and all its derivatives, had no effect on CK19+ cell count, indicating that their effects were restricted to hepatocyte differentiation, as they did not influence cholangiocyte differentiation. Moreover, these effects were observed at low concentrations, similar to plasma concentrations obtained after a single oral administration [To clarify the mode of action of GL, we investigated the structure D and actstration . Mono ex3.3O-glycosides, and found that Mono exhibited the highest sweetness level. The relationship between structure and sweetness found in their study is similar to the relationship between structure and haptic differentiation activity found in this study. Actually, GL has been found to stimulate the sweet receptor, T1R3, in pancreatic β-cells [+ cell count  and significantly decreased albumin+ cell count at a concentration of 500 μM in a dose-dependent manner . Treatment with 50 μM sucralose significantly increased albumin+ cell count . Thus, these results strongly suggest that the hepatocyte differentiation effects of GL involve in T1R3.Mizutani et al.  investig β-cells . Therefo β-cells  and agon β-cells , respect3.4+ cell count . Although there is no significant difference in Numb+ cell count between Mono treatment and control , it increased by Mono treatment. In contrast, Notch+ cell count was not affected; however, the same treatment significantly decreased Hes5+ cell count . Thus, Mono, a GL derivative, was found to stimulate canonical Wnt signaling and to suppress Notch signaling during hepatic differentiation.As mentioned above, GL promoted hepatic differentiation but not cholangiocyte differentiation. The fate of hepatoblasts whether they be hepatocytes or cholangiocytes is determined by Notch signaling. Notch signaling and its downstream transcription factors, Hes1 and Hes5, are crucial for cholangiocyte differentiation ,9,22. Re4In this study, we clarified that GL and some of its derivatives promote hepatic differentiation. At present, GL has been shown to have various effects on liver disease, including anti-oxidative stress, anti-inflammatory and immunoregulatory, anti-viral, anti-steatosis, anti-liver fibrosis and anti-cirrhosis, and anti-cancer effects ,. AlthougAs shown in The relationship between structure and sweetness  is simil+ cell count, and decreased Hes5+ (a Notch target) cell count. These results indicate that Mono activates canonical Wnt signaling and suppresses Notch signaling, which is required for cholangiocyte commitment. Ahmad et al. [There are 2 predominant cell types in the liver, hepatocytes and cholangiocytes. The fate of these 2 cell types depend on Wnt and Notch signaling . During d et al.  reportedd et al.  reportedd et al.  showed tThe authors report no conflicts of interest.


The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function. Summary: Nine amino acids suppress CSF1-stimulated macropinocytosis by promoting the loss of CSF1 receptor, indicating a regulation by nutrients of growth factor receptor signaling. Macropinocytosis is an actin-driven cellular process in which cells internalize relatively large volumes of fluid into plasma membrane-derived vesicles known as macropinosomes. It has been implicated in antigen presentation, cell growth and metabolic regulation . MacropiMacropinocytosis provides a mechanism for nutrient uptake that can support growth for tumor cells and lymphocytes . FollowiDictyostelium, arginine, lysine and glutamate can individually upregulate macropinocytosis .We first examined the effect of leucine on macropinocytosis, as leucine has a well-characterized role as an activator of mTORC1 . BMM werIn dendritic cells, FDx endocytosis is mediated in part by the mannose receptor . In BMM,We next asked whether other amino acids could suppress macropinocytosis. We performed similar experiments as in Combined suppressors and non-suppressors behaved like the individual amino acids: macropinocytosis was suppressed in cells incubated with the nine suppressive amino acids but was not suppressed when incubated with the 11 non-suppressive amino acids E. HoweveTo examine whether this amino acid-dependent suppression of uptake is present in other forms of endocytosis, we used leucine as a model suppressive amino acid and evaluated its effect on receptor-mediated endocytosis. We measured uptake of fluorescent acetylated low density lipoprotein (AcLDL), which binds to class A scavenger receptors and is internalized in clathrin-coated pits . Cells wTo begin to define a mechanism by which leucine suppresses IL34- and CSF1-stimulated macropinocytosis, we measured the effects of amino acids on cell surface expression of CSF1R. Compared with the PBS control, the suppressors leucine, phenylalanine and isoleucine significantly reduced the cell surface levels of CSF1R. The non-suppressors serine, asparagine and glutamate did not A. As a cTo determine whether CSF1R was sequestered or degraded in response to suppressive amino acids, BMM were incubated in PBS with or without a suppressor , or a non-suppressor . Cells were then fixed, permeabilized and stained to detect both intracellular and surface CSF1R. As a control for CSF1R degradation, cells were incubated in PBS+CSF1, as CSF1 promotes the internalization and degradation of CSF1R . Cells wTo test the hypothesis that CSF1R was being degraded, we incubated cells with leucine in the presence of bafilomycin A1 (Baf). Baf is macrolide antibiotic which functions as a potent inhibitor of the vacuolar ATPase, preventing lysosomal acidification and acid hydrolase-dependent protein degradation . If leucAs leucine appeared to promote neither sequestration nor degradation of CSF1R, we hypothesized that leucine was promoting the release of CSF1R from the macrophages. To test this, we assayed for the presence of CSF1R in the supernatants of cells incubated in either PBS alone, PBS with the suppressor leucine or PBS with the non-suppressor serine. Significantly higher levels of CSF1R were detected in the supernatant of cells incubated in leucine compared with those incubated in serine or PBS alone C-E. ThusTo interrogate the dynamics of CSF1R release, we measured CSF1R levels of cells incubated in PBS with or without leucine at varying times from 1 min until 60 min. We also incubated cells in PBS with CSF1 to compare these dynamics with a degradative process. As expected, cells incubated in PBS alone exhibited constant levels of CSF1R throughout the time course, whereas incubation with CSF1 caused rapid internalization and degradation of CSF1R. In contrast, cells incubated in leucine maintained steady levels of CSF1R for the first 15 min of incubation, followed by a slight decrease from 15 min to 30 min, and finally a large decrease from 30 min to 45 min . CSF1R lFig. S2). Thus, macrophages form smaller macropinosomes when CSF1R levels are decreased by exogenous molecules.Lastly, to identify the mechanism of reduced solute accumulation by suppressive amino acids, we examined cells microscopically in conditions with or without suppressive amino acids. In live cell imaging of cells incubated in CSF1 or CSF1+leucine, we could not discern any obvious differences in ruffling or the process of macropinosome formation. This was likely due to the wide range of morphologies that characterize macropinocytosis . InsteadThe modulation of CSF1R expression described here suggests roles for amino acids in the regulation of macrophage physiology. CSF1R signaling is crucial for macrophage proliferation, survival and differentiation . Many exThe secretion of surface receptors in microvesicles has been shown in the context of an oncogenic form of the epidermal growth factor receptor, EGFRvIII, and of CCR5, the co-receptor for HIV-1 . With reIn vivo, an imbalance of suppressive and non-suppressive amino acids in different nutrient environments, such as nutrient-replete healthy tissue or nutrient-deficient tumor microenvironments, could regulate surface expression of CSF1R, with consequent effects on macrophage growth, differentiation and function. Tumor-associated macrophages (TAMs) are anti-inflammatory and tumor promoting  kinase senses uncharged tRNA molecules, which are abundant under amino acid starvation, and in turn phosphorylates eIF2α, resulting in a global downregulation of protein synthesis , and an SGK (PkbR1), were shown to regulate macropinosome size, with PkbA/PkbR1 double knockouts forming smaller macropinosomes  for immunofluorescence and flow cytometry, Phalloidin-iFluor 488  and recombinant anti-CSF1 receptor antibody  for western blotting were purchased from Abcam. CXCL12 was purchased from Peprotech. LPS from Salmonellatyphimurium was purchased from List Biological Laboratories. We purchased 35 mm dishes with 14 mm coverglass from MatTek Corporation. IRDye 680RD goat anti-rabbit IgG (#926-68071) and IRDye 800CW goat anti-mouse IgG (#926-32210) secondary antibodies for western blotting were purchased from LI-COR Biosciences and were diluted 1:10,000.RPMI-1640, fetal bovine serum , GlutaMAX, penicillin-streptomycin (P/S), 70 kDa FDx, LY CH, DiI-AcLDL, unlabeled AcLDL, BSA, goat serum, DAPI solution , paraformaldehyde, 0.25% Trypsin-EDTA with Phenol Red, Prolong™ Diamond Antifade Mountant, DPBS, Hoechst 33342, CellTracker™ Red CMTPX Dye, Halt™ Protease Inhibitor Cocktail and phycoerythrin CXCR4 antibody  were purchased from Thermo Fisher Scientific. IL34 and recombinant mouse CSF1 were purchased from R&D Systems. HEPES, 2-mercaptoethanol, sucrose, Amicon Ultra-2 ml centrifugal filters and all amino acids were purchased from Sigma-Aldrich. Baf from 6 cells/ml were frozen in the culture media described above with 10% DMSO and stored in liquid nitrogen. All animal-related procedures were approved by the University of Michigan Committee on Use and Care of Animals.Macrophages were generated from C57BL6/J mice (Jackson Laboratory). Both male and female mice between the ages of 3 and 12 months were used. Bone marrow flushed from mouse femurs was differentiated into macrophages by culturing for 5 days in RPMI supplemented with 20% FBS, 50 ng/ml recombinant CSF1, 1% glutamax, 0.1% P/S and 37 µM 2-mercaptoethanol. Macrophages were detached using cold PBS lacking calcium and magnesium, and then 3-4×10PBS used for all cell incubations contained the following ingredients: 0.90 mM calcium chloride, 0.49 mM magnesium chloride, 2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic, 137.93 mM sodium chloride, 8.06 mM sodium phosphate dibasic, containing 15 mM HEPES buffer (pH 7.2). For macropinocytosis assays, 1E6 BMM were plated on 60 mm treated dishes in RPMI containing 10% FBS, 1% glutamax, 0.1% P/S, 50 ng/ml CSF1 and 37 µM 2-mercaptoethanol. The medium was replaced with fresh medium 4 h after seeding. At the end of the following day, the medium was aspirated and replaced with RPMI containing 10% FBS, 1% glutamax and 0.1% P/S. The following day, cells were incubated with 300 ng/ml CSF1 and 0.5 mg/ml FDx or 0.5 mg/ml LY. For LPS treatments, cells were pretreated with 100 ng/ml LPS for 30 min, followed by addition of FDx. For PMA treatments, cells were pretreated for 15 min with 100 nM PMA, followed by addition of FDx. For IL34 and CXCL12 treatments, cells were treated with FDx and 100 ng/ml IL34 or 50 nM CXCL12.For flow cytometry-based measurements of cell surface receptor levels, cells were plated on 60 mm untreated dishes in RPMI containing 10% FBS, 1% glutamax, 0.1% P/S, 37 µM 2-mercaptoethanol and 300 ng/ml CSF1. The medium was replaced with fresh medium 4 h after seeding. The experiments were performed the following day. Cells were stained with either 10 ng/µl anti-CSF1 receptor antibody or anti-CXCR4 antibody. For assays examining receptor-mediated endocytosis, cells were incubated for 15 min with 5 µg/ml DiI-AcLDL with or without 50 µg/ml unlabeled Ac-LDL, followed by rinsing and analysis by flow cytometry.For western blot experiments, 2E6 BMM were seeded in 60 mm tissue culture-treated dishes in RPMI containing 10% FBS, 1% glutamax, 0.1% P/S, 37 µM 2-mercaptoethanol and 50 ng/ml CSF1. The medium was aspirated and replaced with fresh medium 4 h later. At the end of the following day the medium was aspirated and replaced with RPMI containing 10% FBS, 1% glutamax and 0.1% P/S overnight. For experiments involving Baf, the overnight medium was aspirated and replaced with fresh medium containing 500 nM Baf. For conditions not receiving Baf, the medium was replaced with fresh medium. Cells were pre-treated in Baf for 60 min followed by another 60 min in the assay conditions.For measuring macropinocytosis, BMM were seeded in 60 mm tissue culture dishes at 1E6/dish. On the day of the experiment, cells were washed 4× with PBS, then incubated in PBS containing 0.25 mM of the specified amino acid(s) or in buffer alone for 30 min at 37°C. Then 70 kDa FDx and either CSF1, LPS, CXCL12, IL34 or PMA were added for 60 min at 37°C. To remove cells from the dish, 0.25% trypsin-EDTA was added to the cells for 3 min at 37°C, at which point RPMI containing 10% FBS was added to the cells. Cells were removed from the dish by gentle scraping.g for 10 min and then resuspended in 100 µl of PBS. Then 1 µg of anti-CSF1 receptor antibody or anti-CXCR4 antibody was added to the cells.For measuring cell surface receptor levels, BMM were seeded in untreated 60 mm culture dishes at 2E6/dish. To begin the experiment, cells were washed 4× with PBS, and then incubated for 60 min in PBS containing 0.25 mM of the specified amino acid, or in PBS alone at 37°C. Cells were removed from the dishes by gentle scraping. Cells were pelleted by centrifugation at 500 For measuring receptor-mediated endocytosis, BMM were seeded in 60 mm tissue culture dishes at 1E6/dish. On the day of the experiment, cells were washed 4× with PBS, and then incubated for 30 min at 37°C in PBS only, or PBS containing 0.25 mM of the specified amino acid. Following this, cells were incubated with 5 µg/ml DiI-AcLDL, with or without 50 µg/ml unlabeled AcLDL, for 15 min at 37°C. Cells were washed with PBS. To remove cells from the dish, 0.25% trypsin-EDTA was added to the cells for 3 min, followed by the addition of RPMI containing FBS. Cells were removed from the dish by gentle scraping. All flow cytometric analysis was carried out using either a BD LTRFortessa or BD Canto (Becton-Dickenson).2), temperature-controlled stage set at 37°C and a DAPI-FITC-Texas Red dichroic mirror (Chroma Technology). For each field of view, phase-contrast, 400 nm excitation 455 nm emission, and 490 nm excitation 535 nm emission images were taken using Metamorph Image Analysis Software (Molecular Devices).For quantifying macropinosome area and number in live cells, 6E4 BMM were seeded in a MatTek dish and cultured as detailed above. Cells were washed 4× with PBS and incubated in PBS containing 0.25 mM of the specified amino acid or PBS alone for 30 min at 37°C. CSF1 and FDx were added to the cells for 5 min, after which the cells were washed with PBS, placed in PBS containing Hoechst 33342 (1000×) and imaged for 4 min. Images were acquired on a Nikon TE300 inverted microscope equipped with a mercury arc lamp, Plan-Apochromat 60×, 1.4 NA objective, cooled digital CCD camera . Cells were then washed, mounted and imaged 48 h later using a Nikon X1 Yokogawa Spinning Disk Confocal microscope equipped with an iXon Ultra 888 camera, with Plan Apo 100×/1.45 oil objective.For quantifying macropinosome area and number in fixed cells, 1E6 BMM were seeded on 18×18 mm glass coverslips placed in a 35 mm dish and cultured as detailed in the text. Following the 30 min incubations, the media was aspirated and replaced with PBS containing FDx, Hoechst 33342 and CSF1. Following a 5 min pulse, cells were fixed using fixation buffer 1 [20 mM HEPES (pH 7.4), 2% paraformaldehyde, 4.5% sucrose, 70 mM NaCl, 10 mM KCl, 10 mM MgClFor quantifying CSF1 receptor levels, 1E6 BMM were seeded on 18×18 mm glass coverslips placed in a six-well plate. The cells were cultured in the same manner as for standard macropinocytosis assays. Cells were incubated for 60 min in their respective conditions and then fixed for 15 min using 4% paraformaldehyde in PBS. The cells were then washed for 15 min using PBS (containing 0.1% Triton X), then incubated for 45 min in blocking buffer . CSF1R antibody, DAPI and Fluorescein-Phalloidin dyes were then added for 30 min at room temperature. Cells were washed and mounted in Prolong Diamond, then imaged at least 48 h later using a Nikon X1 Yokogawa Spinning Disk Confocal microscope equipped with an iXon Ultra 888 camera, with Plan Apo 100×/1.45 oil objective.https://imagej.nih.gov/ij/docs/menus/analyze.html#gels).For performing western blots on cell lysates, medium was aspirated and 100 µl of lysis buffer (1% NP-40 lysis buffer with 1× complete mini protease inhibitor) was added to the cells. Cells were scraped, collected and incubated on ice for 15 min. After centrifugation, 4× Laemmli buffer with β-mercaptoethanol was added to the supernatant. Cell lysates were separated by SDS-PAGE, and protein was transferred to a nitrocellulose membrane by a semidry transfer method. The membrane was blocked with blocking buffer (5% BSA w/v and 0.1% Tween-20 v/v in PBS) for 30 min at room temperature. Primary antibodies diluted in blocking buffer were added at 4°C overnight. Membranes were washed with PBS and then incubated with secondary antibodies  in blocking buffer for 30 min, followed by a wash in PBS. Western blots were visualized using the LI-COR Odyssey infrared imaging system. Gels were quantified according to the ImageJ densitometric gel analysis protocol for 1D gels  was first added to the collected supernatants, which were then spun at 1000 github.com/zmendel/Joel-Swanson-Lab). In brief, single cells were defined by propagation of nuclear objects based on Hoechst 33342 staining to the cell periphery as defined by ilastik cell masks. Macropinosomes were defined based on object segmentation of intracellular FDx signal. Macropinosome number and area were measured and related to individual cells. The pipeline was validated using the control conditions (±CSF1) before being blindly applied to the remainder of experimental conditions. The average frequency and size of macropinosomes per cell from at least four experiments are reported for each condition.To measure the frequency and size of macropinosomes on a single-cell basis using microscopy, we developed an automated image analysis pipeline which uses the open-source software ilastik™ and CellProfiler™. Phase contrast images were processed to generate cell masks using the ilastik Pixel Classification workflow. This assigns the probability that pixels in an image fit user-defined criteria in a Random Forest machine learning model. In these experiments, 10% of images were used for training, sampling 3-4 cells and 3-4 background regions in each image, and the remaining 90% of images were automatically analyzed. ilastik probability maps were exported as cell masks. Finally, a CellProfiler pipeline was developed to quantify the number and size of macropinosomes per cell. The CellProfiler pipelines used for this study are available at GitHub . The average intensity of CSF1R on a per cell basis was quantified. The pipeline was validated using the control condition (+CSF1).t-tests for all experiments comparing the experimental condition with the PBS control. For experiments showing data relative to the PBS condition, statistics were applied using the raw values as opposed to the relative values, which are shown. P-values less than 0.05 were considered significant .Statistical analysis for all experiments was performed using GraphPad Prism software. At least three independent experiments were performed in all cases using cells from at least two different mice. In each graph, bars indicate mean±s.e.m. Analysis was carried out using two-tailed ratio paired