Cell lines are extremely useful for both basic and clinical research. Thus, establishing endometrial cancer cell lines with malignant histology is important. This study aimed to extensively characterize an endometrial clear cell carcinoma cell line.This cell line, named 150,057, was derived from the endometrial clear cell cancer of a 63-year-old woman. The morphology, chromosomes, chemosensitivity, tumor markers, xenotransplantation characteristics, and cancer-related genes of the cell line were characterized.FGFR3 and ARID1A.This cell line exhibited adequate growth, being passaged more than 70 times. The morphology of the cells was polygonal with a cobblestone-like appearance. Karyotyping of the cell line revealed a hypodiploid chromosomal number. 150057 cells expressed CA19–9 and CA125. The cell line was sensitive to doxorubicin, paclitaxel, carboplatin, and cisplatin. After the cells were transplanted into the subcutaneous region of non-obese diabetic-severe combined immunodeficiency mice, they generated xenograft tumors with similar histology as the original tumor. A total of 59 somatic nucleotide mutations were identified in 25 of the 53 examined tumor suppressor genes and oncogenes. Two novel mutations were found in We established and characterized an endometrial clear cell carcinoma cell line that may be useful in carcinogenesis and treatment research for endometrial cancer.Supplementary information accompanies this paper at10.1186/s12885-020-07567-w. Cancer is the second leading cause of death globally . EndometHistologically, endometrial cancer is divided into two categories . Type 1 A unique cancer cell line is important for the progress of cancer research. It can be used to study cancer treatment mechanisms, dosage, toxicity, and other factors. According to previous reports, 24 endometrial cancer cell lines are available , 9, noneTherefore, this study aimed to characterize a new endometrial CCC cell line derived from a patient with cancer.The patient was a 63-year-old woman who was diagnosed with endometrial cancer several years ago. Her tumor marker levels were elevated . We performed a laparoscopic abdominal total hysterectomy, bilateral salpingo-oophorectomy, bilateral pelvic lymph node dissection, and omentectomy. Histology revealed mixed high-grade clear cell carcinoma and grade 1 endometrioid adenocarcinoma of the endometrium. The level of myometrial invasion was less than 50%. No regional lymph node metastasis or lymphovascular invasion was noted. The FIGO stage was IA. We obtained informed consent from the patient before surgery. The entire study protocol was approved by the institutional review board . We confirmed that all experiments were performed in accordance with relevant guidelines and regulations.After surgery, she received adjuvant chemoradiation with cisplatin to treat high-grade CCC. After treatment, the tumor markers regressed to normal levels.We used 10% formalin  to fix the tumor sections, followed by embedding in paraffin. Tissue was cut to a thickness of 4 μm and dewaxed in xylene for 5 min three times followed by 100% alcohol for 5 min, 90% alcohol for 5 min, and 80% alcohol for 5 min. The samples were then washed in PBS (Gibco) for 5 min. Tissues were blocked with hydrogen peroxide for 10 min, followed by three washes in PBS for 3 min each and UV block for 5 min. Samples were then washed with PBS three times for 3 min each. The sections were stained with H&E . Sections were also incubated with primary monoclonal antibodies at 4 °C overnight. Tissues were subsequently incubated with an HRP-linked secondary antibody for 10 min and then with diaminobenzidine tetrahydrochloride  for 5 min to detect reactivity. Slides were counterstained lightly with hematoxylin for 5 min, dehydrated, and mounted in mounting medium . We recorded photographs of the stained sections using a light microscope .Antibodies against WT1  , MIB1 . The membrane was blocked at room temperature in a solution of 3% nonfat dry milk in PBS and 0.1% Tween-20 and then rinsed in PBS/0.1% Tween-20. Blots were incubated with diluted solutions of anti-P53   and anti2) was finely cut into small pieces (1–2 mm3) using no. 15 blades in a 10-cm dish containing Dulbecco’s Modified Eagle’s Medium  without serum. The tissues were dissociated with 0.1 mg of collagenase IA (Sigma-Aldrich) and incubated for 60 min at 37 °C. After enzymatic digestion, the resultant cells were placed in DMEM  supplemented with 10% fetal bovine serum , penicillin, and streptomycin (Gibco). The cells passaged every week.The endometrial tumor (2 × 2 cmwww.atcc.org/STR%20Database.aspx)  to exam the morphology of cancer cells in a culture dish.2 on day 1. The culture medium was changed every 3 days. We harvested the cells on days 7. Doubling time was determined using the equation doubling time = duration × log (2)/log  − log  [Proliferation assays were performed at P10, P26, and P41. We placed cells in 96-well plates  at a density of 3000 cells/cm number) .Aneuploidy is a unique feature of cancer cells . To clarCells were fixed with 4% paraformaldehyde (Sigma) for 10 min, permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, blocked with 4% normal goat serum (Sigma) for 30 min, and then treated with primary antibodies against CD133  and CK7  overnight at 4 °C. Cells were then incubated with fluorescein isothiocyanate  or rhodamine  at a concentration of 1:200 as the secondary antibody for 1 h at room temperature in the dark. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole . The coverslip was mounted with a drop of mounting medium , and cells were observed under a microscope.We used flow cytometry to quantify the proportion of CSCs in 150,057 cells. We detached the cells using PBS containing Accutase . Then, we collected the cell pellet after centrifugation at 1200 rpm for 5 min. Cells were then washed three times with PBS. Thereafter, we incubated the cells with the target antibodies  conjugated with FITC or phycoerythrin  for 30 min. After washing cells three times with PBS, cells were analyzed using a flow cytometer .6 cells/5 mL were cultured for 7 days to examine tumor markers including CA125, CA 199, Carcinoembryonic antigen (CEA), HCG, and squamous cell carcinoma antigen (SCC) antigen. All ELISA kits were purchased from Thermo Fisher Scientific.ELISA was performed to assess tumor marker expression. In total, 2 × 10The current chemotherapy regimen for CCC includes doxorubicin, carboplatin, cisplatin, and paclitaxel . The eff5 150,057 CD133+ (P10) cells subcutaneously into their backs. We raised these mice in a pathogen-free room at a temperature of 22 °C and a relative humidity of 30–70% at the animal center in Tzu Chi University. The personnel in the animal center were in charge of their feeding. The development of a tumor was confirmed, and the tumor size and weight were measured. After 12 weeks, at which point the tumor reached 5–10 mm in diameter, the mice were euthanized with CO2 and followed by cervical dislocation. Then the tumor was removed. Each xenograft was processed for histological examination. The tumor slices were fixed in 10% formalin and embedded in paraffin. H&E staining was used to observe tumor morphology. Immunohistochemistry was used to observe specific protein expression. The Hualien Tzu Chi Hospital Animal Use Protocol Board provided full approval for this research (No. 104–05-01).Three female 6-week-old NOD-SCID mice  were obtained from Tzu Chi University and inoculated with 1.5 × 10To assess specific cancer-associated protein expression, we examined WT1, MIB1, Annexin IV, ER, and PR in xenografts. The specimens in the paraffin block were cut to a thickness of 4 μm and deparaffinized. Then, the slices were stained with antibodies  as described previously. DAB was then used to detect reactivity. We captured the images using a light microscope (Nikon).ARID1A, CCNE1, and PPP2R1B [To perform mutational analysis, we extracted genomic DNA from 150,057 cells (P24) using a Qiagen kit  following the manufacturer’s instructions. After the quality and quantity assessment, NGS was performed according to the instructions of Illumina (TruSeq Enrichment guide) . Briefly PPP2R1B –27. The A tumor was excised from a 63-year-old woman with endometrial cancer. To clarify the histology expression status of the primary tumor, we stained the specimen with H & E staining. The original tumor was mixed endometrioid and clear cell carcinoma  can cause tumor chemoresistance and recurrence. Therefore, we next identified the CSC population in 150,057 cells using immunofluorescence and flow cytometry. CD133 was used as a CSC marker, and EPCAM and cytokeratin 7 (CK7) were used as epithelial cell markers.Immunofluorescence revealed positive staining for CD133 and CK7 (a mullerian origin marker)  in 150,057 cells were 0.7 μg/mL for doxorubicin Fig. a, 323.7 To determine tumor marker expression in the 150,057 cells, we collected conditioned medium from 150,057 cells and used Enzyme-linked immunosorbent assay (ELISA) to evaluate tumor marker levels. Positivity was noted for CA125 (1091.6 U/mL) and CA19–9 (65.9 U/mL), whereas CEA, human chorionic gonadotropin (HCG), and SCC antigen expression was not detected  mice as a xenograft model for 150,057 cells. The results illustrated that xenografts were generated in all three examined mice after the injection of CD133+ 150,057 cells, and the average tumor size was approximately 2 × 1 cmImmunohistochemistry of the original and xenografted tumor revealed positivity for WT1 Fig. a, f, MIBWe used Western blot to detect another two proteins P53 and HNF1β which will be expressed in endometrial CCC to avoid non-specific binding of the proteins in immunohistochemistry. Figure TP53 and PIK3R1 in endometrial cancer are correlated with tumor staging and survival [FGFR3 and ARID1A were not reported in dbSNP and Catalog of Somatic Mutations in Cancer database  is depleted [ARID1A causes chromatin remodeling dysfunction, which changes the expression of multiple genes including CDKN1A, SMAD3, MLH1, and PIK3IP1, thereby contributing to carcinogenesis [ARID1A gene in endometrial CCC.A high frequency of mutations is observed in d PIK3CA , 40. In depleted . Mutatioogenesis . These cARID1A mutation and PI3K/AKT pathway activation [PIK3CA mutation and resistance to mTOR inhibitors [ARID1A-mutated ovarian CCC cells [ARID1A may represent a new target for the treatment of endometrial CCC.ARID1A is a subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, which is related to cell cycling and proliferation . The SWItivation . At prestivation . The mulhibitors . EZH2-taCC cells . Taken tFGFR3 mutation has been found in two-thirds of low-grade papillary bladder cancers [FGFR3-TACC3 fusion (potent oncogenes) has been reported to result in a related metabolic disturbance in glioblastoma and gastric cancer. FGFR3 mutation is also correlated with poor prognosis in oral cancer [FGFR3 mRNA overexpression is linked to poor prognosis in colon cancer [FGFR3 is one of the 4 FGFR tyrosine kinases (FGFR1–4) . When FG cancers . FGFR3-Tl cancer . FGFR3 mn cancer . In our FGFR3 mutation in relation to therapeutic response, a previous study illustrated that the FGFR3-targeting drug anlotinib has better treatment efficacy in endometrial cancer than conventional chemotherapy with carboplatin and paclitaxel [FGFR-mutated endometrial cancer reported an overall response rate (ORR) of 9.4%, a partial response rate of 9.4%, progression-free survival (PFS) of 3.3 months, and overall survival (OS) of 10.1 months [FGFR-mutated endometrial cancer reported ORRs of 14.3–21.8%, PFS of 5.4 months, and OS of 10.6 months [FGFR1–4 mutations [ARID1A and FGFR3, and for examining the involvement of ARID1A and FGFR3 mutation in the development, survival, and progression of cancer.Regarding clitaxel . A phase1 months . A separ6 months . In addiutations . Taken tIn conclusion, we successfully derived a unique endometrial CCC cell line from a patient for use in future cancer research. Specifically, the cell line can be used for further endometrial CCC research to clarify the pathogenic mechanism and improve treatment.Additional file 1: Supplement Table 1. Profiling of short tandem repeat in 150,057 cells and DNA, and the comparison of the ATCC STR database.Additional file 2:.


Citrus peel degreening is promoted by low temperature via modulation of multiple genes associated with chlorophyll degradation, carotenoid biosynthesis, photosystem disassembly, phytohormones, and transcription factors without involving ethylene signalling. Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5–20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit ( Fruit ripening is a multifaceted process comprising various physiochemical and structural changes such as softening, degradation of starch to sugars, development of colour, and production of aroma volatiles . In citrThere are two main pathways that have been linked to citrus peel degreening. The first is chlorophyll degradation, which initially involves dephytilation of chlorophyll molecules by chlorophyllase (CLH) and pheophytinase (PPH), followed by removal of the central Mg atom by Mg-dechelatase to form pheophorbide. Pheophorbide is then converted to red chlorophyll catabolites (RCC) by pheophorbide a oxidase (PaO), and RCC is reduced to colourless compounds by RCC reductase (RCCR)  in several citrus fruit species. Typically, the seasonal temperature drops as most citrus fruit mature on the tree. Previous reports have correspondingly demonstrated that cold periods below 13 °C are required to stimulate on-tree fruit colour development  compared to 25 °C. RNA-seq unveiled a distinct set of genes that were differentially regulated by low temperature independently of ethylene. Furthermore, repeated treatments of fruit with 1-methylcyclopropene (1-MCP), a well-known ethylene antagonist , failed Citrus limon L. cv. ‘Allen Eureka’) grown under standard cultural practices were collected in 2018 from a commercial orchard in Takamatsu, Kagawa, Japan. To examine on-tree degreening, sampling took place at seven time-points during fruit development on 3 Sept., 27 Sept., 12 Oct., 30 Oct., 14 Nov., 29 Nov., and 13 Dec., corresponding to 171, 196, 211, 230, 246, 261, and 276 d after full bloom (DAFB), respectively. To characterize postharvest effects of ethylene, fruit at 196 DAFB were divided into four groups containing 20 replicates each. The first group was used as the untreated control, the second group was treated with 2 µl l–1 1-MCP for 12 h, the third group was continuously treated with 100 µl l–1 ethylene, while the fourth group was initially treated with 2 µl l–1 1-MCP for 12 h followed by continuous treatment with 100 µl l–1 ethylene. 1-MCP was released by dissolving SmartFresh™ powder  in water. All treatments were carried out at 25 °C for up to 8 d. For postharvest storage tests, fruit at 196 DAFB were divided into five groups of 40 replicates each, and stored at either 5, 10, 15, 20, or 25 °C for up to 42 d. In addition, three separate groups of 40 fruit each were stored at either 5, 15, or 25 °C with 1-MCP treatments for 12 h repeated twice a week. Samples of fruit peel (flavedo) were collected, frozen in liquid nitrogen, and stored at –80 °C for future analysis. At each sampling time, the flavedo was collected from each of three replicate fruits.Lemon fruit , N6-isopentenyladenine (iP), and salicylic acid (SA), and 13C-labelled jasmonoyl-L-isoleucine (JA-Ile). Eluted fractions were analysed on a 1260–6410 Triple Quad LC/MS system equipped with a ZOR-BAX Eclipse XDB-C18 column (Agilent Technologies). Liquid chromatography conditions are described in JXB online, and the multiple-reaction-monitoring mode of the tandem quadrupole MS and precursor-product ion transitions for each compound are listed in Phytohormone extraction and analysis were performed according to the method described by ® Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs), before being sequenced using an Illumina HiSeq 2500 platform . Trimming was done to obtain ≥10 million paired reads per sample, and the reads were mapped to the reference Citrus clementina Genome v1.0  was used as the housekeeping gene after examining its constitutive expression pattern in the RNA-seq results. Relative expression values were calculated using fruit at 196 DAFB (0 d).Total RNA was extracted from the flavedo of fruit at the time of sampling from the tree , after 4 d for the ethylene, 1-MCP+ethylene, and control groups, and after 28 d storage at either 5, 10, 15, 20, or 25 °C. To remove genomic DNA contamination from the extracted RNA, treatment with DNase I  was carried out, followed by clean-up with FavorPrep after using a Tri-Reagent RNA Clean-up Kit . For all treatments, 2.4 µg of clean RNA was reverse-transcribed to cDNA using a TaKaRa RNA PCR™ kit. Gene-specific primers were designed using the Primer3 software . Differences in CCI, pigment and phytohormone contents, and gene expression were determined using ANOVA followed by Tukey’s post hoc test.Data were subjected to statistical analysis using R v.3.4.0 , and pre-treatment with 1-MCP completely abolished this effect , whereas no significant changes were observed at 25 °C  that were regulated by either ethylene or low temperature  were ‘metabolic process’ and catalytic activity’ . There wClCLH1 and ClPPH) have been previously associated with chlorophyll degradation in citrus fruit , for validation and further analysis by RT-qPCR, and the results confirmed that both ethylene treatment and low temperature caused a reduction in expression levels , which is associated with ABA biosynthesis, for additional analysis by RT-qPCR. The results confirmed that it was up-regulated both after 4 d of ethylene exposure and 28 d of storage at low temperatures  using repeated 1-MCP treatments were unsuccessful  complex , the MYB, bHLH, and bZIP gene families are associated with chlorophyll metabolism while NACs and ERFs regulate carotenoid biosynthesis , bHLHs (AtPIF4 and AtPIF5), and ethylene insensitive 3 (AtEIN3) have been shown to significantly enhance leaf senescence by promoting the activity of genes related to chlorophyll degradation (ynthesis . Thus, eClERF114  and sharClbHLH25 , whereas (ClERF3 , as wellClbHLH25 .ClCLH1 and ClERF114, which we identified as exhibiting an ethylene-specific pattern of expression (ClCLH1 homologues in other citrus fruit exhibit a dramatic induction in response to ethylene treatment and yet lack a measurable increase in expression during natural degreening (ClCLH1 was only up-regulated in the presence of ethylene (ClPPH, ClLHCB2, ClPSY1, ClLCYb2a, ClCHYb1, ClERF3, and ClbHLH25, also exhibited similar expression patterns during on-tree maturation (In our study, low temperature also appeared to play a prominent role in natural peel degreening during on-tree maturation of the lemon fruit. Peel degreening and the associated reduction in the content of chlorophylls and carotenoids coincided with the gradual decline in minimum ambient temperatures to below 13 °C . Previoupression , 8B, didpression , F. Prevethylene . In contturation , indicatIt is also important to note that many genes were differentially expressed in fruit at 5 °C and yet the rate of peel degreening was significantly slower than in fruit at 10–20 °C. This was probably due to low activity of the enzymes associated with degreening at 5 °C, as low temperature is known to generally decrease enzyme activity in plants (The regulation of fruit ripening by low temperature is not unique to citrus fruit. Previous studies have also demonstrated its role, either independently or in concert with ethylene, in the regulation of fruit ripening in various species such as kiwifruit a, 2019b,Based on the results of this study, we present a simplified model that summarizes the distinct ethylene-dependent and low temperature-modulated regulatory mechanisms of peel degreening in citrus fruit . AccordiFig. S1. Chromatogram showing the carotenoids identified in the peel of lemon.Fig. S2. Selected GO terms enriched among DEGs responding to ethylene, and storage at 5 °C and 15 °C.Table S1. Liquid chromatography conditions for phytohormone analysis.Table S2. Parameters used for LC-ESI-MS/MS analysis of phytohormones.Table S3. Primer sequences used for RT-qPCR.Table S4. DEGs exclusively responding to ethylene.Table S5. DEGs responding to ethylene or to low temperature at 5 °C.Table S6. DEGs responding to ethylene or to low temperature at 15 °C.Table S7. DEGs responding to either ethylene, or to low temperatures at 5 °C or 15 °C.Table S8. DEGs exclusively responding to low temperature at 5 °C.Table S9. DEGs responding to low temperature at either 5 °C or 15 °C.Table S10. DEGs exclusively responding to low temperature at 15 °C.Table S11. Selected DEGs associated with peel degreening.eraa206_suppl_Supplementary-Figures-S1-S2Click here for additional data file.eraa206_suppl_Supplementary-Tables-S1-S2Click here for additional data file.eraa206_suppl_Supplementary-Tables-S3-S11Click here for additional data file.


In vivo 125c was considerably less toxic (LD50 1705 mg/kg) than benzalkonium chloride, miramistine, and chlorhexidine at oral administration on CD-1 mice. An aqueous solution of 125c (0.2%) was shown to be comparable to reference drugs efficiency on the rat’s skin model. The molecular target of 125c seems to be a cellular membrane as other quaternary ammonium salts. The obtained results make the described quaternary bis-ammonium pyridoxine derivatives promising and lead molecules in the development of the new antiseptics with a broad spectrum of antimicrobial activity.A series of 108 novel quaternary bis-ammonium pyridoxine derivatives carrying various substituents at the quaternary nitrogen’s and acetal carbon was synthesized. Thirteen compounds exhibited antibacterial and antifungal activity (minimum inhibitory concentration (MIC) 0.25–16 µg/mL) comparable or superior than miramistin, benzalkonium chloride, and chlorhexidine. A strong correlation between the lipophilicity and antibacterial activity was found. The most active compounds had logP values in the range of 1–3, while compounds with logP > 6 and logP < 0 were almost inactive. All active compounds demonstrated cytotoxicity comparable with miramistin and chlorhexidine on HEK-293 cells and were three-fold less toxic when compared to benzalkonium chloride. The antibacterial activity of leading compound  The drastic worldwide rise of antimicrobial drug resistance would lead to the death of 10 million people every year by 2050, which becomes a proven and challenging problem ,4. ThereQAC (quaternary ammonium compounds) is one of the best-known group of antiseptics. The prominent representatives of this class are benzalkonium chloride, cetylpyridinium chloride, miramistin, and didecyldimethylammonium chloride ,7. The mSince QAC-based antiseptics could be inefficient because of acquired resistance ,8, have 125c with advantageous antimicrobial properties  derivatives . In prevoperties . These p125c in seven steps with the overall yield of only 13%. Then we developed a new synthetic approach in four steps with an overall yield of 22% ethanesulfonic acid) buffer . Then, 200 μL of suspension were placed into a 96-well plate which were mixed with 10 μM of NPN. The background fluorescence was recorded  by Spectrophotometer Infinite M200 PRO . Next, 20 μL of 125c with final concentrations equal to minimal bactericidal concentration (MBC), 2 × MBC, and 4 × MBC, was added to the E. coli suspension into a 96-well plate. The fluorescence was recorded until no further increase of fluorescence was detected. The NPN uptake percent was calculated using the following formula:The outer membrane permeability was assessed by measuring the fluorescent dye NPN (1-N-phenylnaphthylamine) uptake ,36. Gram125c in MBC/2 × MBC/4 × MBC,Fobs—fluorescence of E. coli in the absence of 125c,F0—the initial NPN fluorescence with E. coli after addition of 10 μg/mL polymyxin B , which was used as a positive control.F100—NPN fluorescence with 3(5), (Sigma-Aldrich). Gram-positive (S. aureus ATCC209p) and Gram-negative (E. coli CDCF-50) bacteria were incubated to a mid-log phase in Mueller Hinton broth , harvested by centrifugation (3.000 rpm for 10 min), washed twice, and diluted to an optical density of 0.05 at 600 nm with 5 mM HEPES buffer  containing 0.1 M KCl to equilibrate the cytoplasmic and external K+. Then, the cell suspensions (180 μL) were mixed with 20 μL of 0.8 μM DiSC3(5) in a 96-well plate and incubated for 15 min in darkness  by Spectrophotometer Infinite M200 PRO . After that, 125c and reference compounds  in 0.5, 1, and 2 × MBCs were added to cell suspensions into a 96-well plate. Changes in fluorescence were observed during 20 min of recording.The cytoplasmic membrane depolarization was determined by using the membrane potential-sensitive fluorescent dye 3,3-Dipropylthiadicarbocyanine Iodide DiSCThe analysis was carried out using a high-resolution scanning electron microscope Merlin . The surface morphology was studied at an accelerating voltage of primary electrons of 5 kV and a probe current of 300 pA for a minimal impact on the study object. Scanning electron microscopy was carried out in the Interdisciplinary center for Analytical Microscopy .125a, 105c, 125c, 105d, 105f, 105g, 105i, 105l, 85m, 105n, 85o, 85p, and 85q) exhibiting comparable or superior antibacterial activity compared to miramistin, benzalkonium chloride and chlorhexidine were identified (MICs 0.25–4 µg/mL on Gram-positive and 0.5–16 µg/mL on Gram-negative bacteria). A strong correlation between the lipophilicity and antimicrobial activity was found. Most active compounds had logP values in the range of 1–3, while compounds with logP > 6 and logP < 0 were almost inactive. In further investigation of antibacterial and antifungal activity on clinical bacterial and fungal isolates, compounds 125c, 105i, 105l, 105n, 85o, 85p, and 85q significantly exceeded miramistin, benzalkonium chloride and chlorhexidine in antibacterial activity. Compounds 105c, 125c, 105d, 105f, 105n, and 85p exhibited a pronounced antimycotic activity than all reference antiseptics against all fungi tested. All active compounds had comparable values of cytotoxicity with miramistin and chlorhexidine and were less toxic when compared to benzalkonium chloride. In vivo, the leading compound 125c was considerably less toxic (LD50 1705 mg/kg) than benzalkonium chloride (LD50 180 mg/kg), miramistine (LD50 1000 mg/kg) and chlorhexidine (LD50 1260 mg/kg) at oral administration on CD-1 mice. Moreover, 125c demonstrated comparable or even higher activity than that of the benzalkonium chloride on biofilm-embedded S. aureus, S. epidermidis, E. coli, and P. aeruginosa. A 0.2% aqueous solution of 125c showed 97.5% growth inhibition of E. coli CD CF-50 after 5 min of exposure on rat’s skin comparable to reference drugs efficiency. The molecular mechanism of antibacterial activity of 125c apparently is the membrane damage since a 125c dose-dependent effect on membrane permeability and polarization was observed. The obtained results allow suggesting described quaternary bis-ammonium pyridoxine derivatives as promising lead molecules for the development of the new antiseptics with a broad spectrum of antimicrobial and antifungal activity.In conclusion, an extended set of 108 novel quaternary bis-ammonium pyridoxine derivatives carrying various substituents at the quaternary nitrogens and acetal carbon have been synthesized. In the course of primary in vitro screening 13 compounds (


Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite  Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis  and has exerted significant impact on African economics, ecosystems, and public health for centuries. Whereas there are currently five drugs for HAT, their applicability depends on disease stage and causative subspecies. Adverse toxicity, complex administration, and emerging resistance all demand new treatments  , in approval and late stage clinical development, respectively 2, 2019) . A secon . A seco2, 2019) 11. IdentT. brucei suramin resistance has been challenging to obtain both in the laboratory or the field beyond a few interesting examples , and lization , but demunlikely . Subsequunlikely , 21. TogIn many organisms, suramin has complex effects, including interactions with phosphatases , the cys50 values of 3–100 μm  and all seven glycolytic enzymes compartmentalized in glycosomes  with IC53–100 μm , 32, sur00 μm  50 ∼3-fold . Silencing reduced accumulation to 0.4 pmol (p < 0.05). ISG65 silencing had no significant effect on suramin uptake compared with WT cells  of the parental cells, respectively  did not significantly affect the abundance of these enzymes (A and B). gPFK and gPGK remain localized to vesicular structures, typical of glycosome staining (C), and following 3–4 days of exposure to suramin, there was no significant alteration. Similarly, there were no changes to cPYK localization.Suramin-treated cells were initially analyzed by immunofluorescence microscopy and immunoblotting. We selected three glycolytic pathway enzymes, two localized within the glycosome and one in the cytosol: phosphofructokinase ) acting before the glycolytic branch point and the most suramin-sensitive of the glycolytic enzymes , phospho enzymes , A and Bstaining C, and foMovie S1 and p = 0.009, n = 10 cells). Total glycosomal volume increased from 0.81 ± 0.22 to 1.079 ± 0.27 μm3 . The volume of individual glycosomes expanded by 12% following 3 days of suramin treatment, with a median volume of 0.0113 μm3 for untreated versus 0.0127 μm3 for treated cells (p < 0.037). To compare individual glycosome volumes, eight untreated and treated cells were analyzed, representing 473 and 592 glycosomes, respectively. Despite most glycosomes retaining volumes within the range of untreated cells, there was a small increase in exceptionally large glycosomes in suramin-treated cells . Further, after 2 h of incubation with 1× or 3× EC50 suramin, mitochondrial membrane potential (MMP) decreased by 10 and 20%, respectively, attaining a ∼50% decrease after 8 h and 3× EC50 suramin (B). In BSFs, the MMP is maintained by the reverse activity of the F1Fo-ATPase complex, which hydrolyzes cytosolic ATP imported into the mitochondrial lumen .To report specifically on mitochondrial and cytosolic ATP levels, BSFs expressing firefly luciferase fused with three C-terminal v5 tags in tandem with or without an N-terminal mitochondrial localization signal (MLS) were generated. The proportions of luciferase present in the cytosol or mitochondrion were verified by immunoblotting . Lucifer13C]glucose after 24-h suramin treatment at the EC50, indicated that 85 metabolites were significantly changed in abundance  and that several essential pathways were impacted. Given that ATP levels and MMP were significantly altered, analysis at 12, 21, and 29 h of suramin treatment was performed to identify and track primary metabolic effects. Both cells and spent culture medium were analyzed, the latter to determine changes to uptake or secretion of specific metabolites. In this second series of experiments, glucose labeling was omitted as 13C isotope distributions were essentially unchanged between suramin-treated and -untreated cell extracts at 24 h, indicating no rapid severe changes to glucose metabolism. From this data set, 48 metabolites, identified against an authentic standard and/or by MS fragmentation pattern, were identified as significantly changed in the cell extracts (Tables S2 and S3).Global metabolomics analysis, labeling cells with suramin, 1 × 107 cells resuspended in uptake assay buffer were incubated with 250 nm [3H]suramin  for preset times up to 60 min. The saturability of uptake was determined by incubation of cells with 250 nm [3H]suramin in the presence of 100 μm unlabeled suramin, which yielded a rate of uptake not significantly different from zero . We observed two phases of suramin uptake: 1) from 0 to 20 min and 2) from 20 to at least 60 min (r2 > 0.97). To compare the rates of suramin uptake for the four cell lines and avoid any early effect caused by the internalization of the drug, 1 × 107 cells of each cell line were incubated with 200 nm [3H]suramin for 15 min, consistently within the first linear phase of transport, in assay buffer only or in the presence of 100 μm unlabeled suramin. The reaction was stopped by the addition of 400 μm unlabeled suramin in uptake assay buffer followed by immediate centrifugation through an oil layer  and 3× EC50 (105 nm) concentrations were monitored using the Molecular Probes ATP Determination Kit , based on the luciferin-luciferase bioluminescent enzymatic reaction. Bloodstream-form cell cultures of T. brucei 2T1 were incubated with and without test compound, and, at each predetermined incubation time, 107 cells of each sample were transferred into a microcentrifuge tube and centrifuged at 2500 × g for 10 min at 4 °C. The pellet was washed twice with 1 ml of 50 mm Tris-HCl (pH 7.4) containing 0.1 mm DTT and resuspended in 150 μl of the same buffer. The samples were incubated with digitonin (40 μm) to facilitate cell membrane permeabilization. Cells were lysed by sonication on ice (twice for 10 s separated by 30 s), using a Soniprep 150 (MSE) at 8-μm amplitude. The samples were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was collected and instantly frozen in liquid nitrogen and stored at −80 °C. Oligomycin (2.0 μg/ml) was used as positive control. ATP levels were quantified using the kit above following the manufacturer's instructions. 90 μl of standard reaction solution was added to each well of a 96-well plate, and the background luminescence was recorded in a FLUOstar OPTIMA fluorimeter; 10 μl of each sample was then added to each well. The plate was incubated at 28 °C for 15 min, and the luminescence was measured, including a standard curve with an ATP concentration ranging from 1 nm to 1 μm to allow the calculation of the ATP concentrations in each sample. The ATP content was measured at 0, 2, 8, and 12 h of incubation with the test sample.Changes in cellular ATP levels due to the exposure of trypanosomes to suramin at 1× ECT. brucei brucei 2T1 were incubated at 1× EC50 (35 nm) and 3× EC50 (105 nm) concentrations of suramin. 3 × 106 cells were harvested at each time point  after further incubation with TMRE (100 nm) for 30 min at 37 °C. The cells were transferred into a microcentrifuge tube and centrifuged at 2500 rpm for 10 min at room temperature. The pellet was washed once in 1 ml of PBS (pH 7.4) and resuspended in 1 ml of fresh PBS. 200 μl of cell suspension was added to each well of a 96-well black flat-bottomed plate, and the fluorescence proportional to the amount of TMRE permeated into mitochondria was recorded in a FLUOstar OPTIMA fluorimeter. Valinomycin  was used as control for mitochondrial membrane depolarization.Changes in mitochondrial membrane potential (Ψm) after incubation of trypanosomes with the suramin were determined using the indicator dye tetramethylrhodamine ethyl ester (TMRE). Cell suspensions of in situ was done spectrofluorometrically using the indicating dye safranin O . T. brucei BSF cells (6 × 107 cells/ml) were resuspended in a reaction buffer containing 8 mm KCl, 110 mm potassium gluconate, 10 mm NaCl, 10 mm HEPES, 10 mm K2HPO4, 0.015 mm EGTA, 0.5 mg/ml BSA (fatty acid–free), 10 mm mannitol, 1 mm MgCl2, 2 mm ATP, and 5 μm safranin O, pH 7.25. The reaction was activated with digitonin (40 μm), whereas oligomycin (3 μm) and SF6847 (250 nm) were injected at specific time points during the assay. Changes in the amount of fluorescence over time were detected on an Infinite M200 microplate reader (TECAN) .Estimation of the ΔΨm in vivo using ATP-dependent luciferase bioluminescence. Briefly, an equal number of BSF cells were treated with suramin at the concentration of 1× EC50 and 3× EC50 for 0, 2, and 12 h, washed with PBS (pH 7.4), and resuspended in HEPES-glu-LUC buffer .The emission of light was triggered by the addition of d-luciferin  and immediately measured on an Infinite M200 microplate reader (TECAN). The cells exhibit a maximum luminescence signal within ∼1 min after d-luciferin addition, after which the signal decayed rapidly; the measurement was taken at peak intensity.Cytosolic and mitochondrial ATP was measured m luciferin A commercial anti-v5-antibody and antibodies recognizing the cytosolic adenosine phosphoribosyltransferase and mitochondrial Hsp70 protein.Expression of luciferase-v5 or MLS_luciferase-v5luciferase in cytosolic and mitochondrial fractions was analyzed, tested by Western blotting using 50 μl-arginine and l-lysine (HMI11-R0K0) or l-arginine U-13C6 and l-lysine 4,4,5,5-2H4 (HMI11-R6K4) (Cambridge Isotope Laboratories) were added at 120 and 240 μm, respectively. Cells treated with 35 nm suramin (1× EC50) were grown in parallel with nontreated cells, in the presence of HMI11-R0K0 or HMI11-R6K4, respectively. Cultures in logarithmic growth phase were mixed after 24 and 48 h, respectively, immediately harvested by centrifugation, washed twice with PBS containing Complete Mini Protease Inhibitor Mixture (Roche Applied Science), resuspended in Laemmli buffer containing 1 mm DTT, and stored at −80 °C. Samples were generated in triplicate, and one label swap was performed. Samples were sonicated, and aliquots containing 5 × 106 cells were separated on a NuPAGE bis-tris 4–12% gradient polyacrylamide gel (Invitrogen). The sample lane was divided into eight slices that were excised from the Coomassie-stained gel, destained, and then subjected to tryptic digest and reductive alkylation. Liquid chromatography tandem MS (LC-MS/MS) was performed by the Proteomic Facility at the University of Dundee. The eight fractions obtained from SDS-PAGE were subjected to LC-MS/MS on an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific). The 24-h suramin treatment set were run on an Orbitrap Velos Pro (Thermo Fisher Scientific). Mass spectra were analyzed using MaxQuant version 1.5 (T. brucei brucei 927 annotated protein database (release 39.0) from TriTrypDB , cultures in logarithmic growth phase were quenched by rapid cooling to 10 °C in a dry ice/ethanol bath, and 108 cells/sample were harvested by centrifugation . Supernatant samples were collected mixed 1:20 with extraction solvent . Cells were resuspended in 1 ml of PBS, transferred to an Eppendorf tube, and washed once with PBS . The pellet was resuspended in 200 μl of extraction solvent, crushed with a pipette tip, and extracted for 1 h at 4 °C under shaking (1300 rpm). After the removal of cell debris by centrifugation , the extract was stored at −80 °C under argon until LC-MS analysis. To assess instrument performance, one pooled quality control sample was prepared by mixing an equal volume of all of the samples.Cultures were grown and treated with 35 net al. (Samples and a mixture of 240 standards (in three mixes) were separated by HPLC on a Dionex Ultimate 3000 RSLC system (Thermo Fisher Scientific) using a ZIC-pHILIC (Merck) column and then analyzed on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) using the method of Rattigan et al. , includiThe standard mixes' raw files where analyzed using ToxID Automated Compound Screening software (Thermo Fisher Scientific). Raw sample data files were converted to mzXML and fragmentation files to mzML using Proteowizard . The fil13C-labeled glucose experiment data set was analyzed using mzmatch-ISO . The time course data set was then analyzed by implementing a linear model using R, followed by analysis of variance to identify metabolites with differential time-dependent changes between treated and untreated cells (model = lm(y ∼ IsTreated*Time), where y is the metabolite intensity). The 65 p values were then corrected using the Benjamini–Hochberg procedure . Data for the 48 significantly changed metabolites where then uploaded to Metaboanalyst (The atch-ISO . The unlPXD014215.All data (except raw 'omics data) are contained within the article. 'Omics data sets for the proteomics and metabolomics analyses are available upon request from the corresponding author. Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository  with the


Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo–DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the “in vivo complex of enzyme” (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis (“comet assay”). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.The benzo[ Especially cytotoxic, but also antimicrobial and anti-inflammatory effects have been previously described [c]phenanthridines was previously optimized into a four step process phenanthridine class, and thus exhibits different biological activities than similar potential chemotherapeutics like indolyl quinoline or acridine derivatives. This might partially be attributed to P8-D6 being more hydrophilic as compared to dual topoisomerase inhibitors already tested in clinical phase I studies. Thus, the compound should be considered a promising alternative to existing dual Topo inhibitors for clinical application. In general, selective inhibition of topoisomerase I results in an increased activity of topoisomerase II and vice versa . Thus, a−1 body weight [The in vivo toxicity study on athymic nude mice indicated reversible toxic effects at a concentration of 5 mg P8-D6 kgP8-D6 was synthesized as recently described . The folv/v) heat-inactivated fetal calf serum and 1% (v/v) penicillin/streptomycin. Media and supplements were purchased from InvitrogenTM Life Technologies . Cell cultivation was performed at 37 °C, with 5% CO2 and under humified conditions. Cells were routinely tested for the absence of mycoplasm contamination. For incubations, P8-D6 was dissolved in DMSO and added to the incubation medium, resulting in a solvent concentration of 1% (v/v) DMSO plus final concentrations of the compound between 0.1 nM and 10 µM.The HT-29 human colon carcinoma cell line was obtained from DSMZ  and grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v DMSO) diluted in live cell imaging solution (LCIS). Subsequently, cells were rinsed with PBS and nuclei counterstained with a “Hoechst 33258” solution containing 10 µL/mL pentahydrate (bis-benzimide) for 20 min. Next, staining solutions were discarded and cells were washed with LCIS. 100 µL/well LCIS was added and fluorescence imaging was performed using DAPI (377.4 nm/447 nm) and GFP (469.5 nm/525 nm) filters with a BioTekTM CytationTM3 Cell Imaging Multi-Mode Microplate Reader and the Gen5 software.In preliminary experiments, P8-D6 was found to possess fluorescent properties at an emission maximum of 530 nm. To confirm the ability of P8-D6 to penetrate cell nuclei, 8000 cells/well were seeded in black 96 well plates and allowed to grow for 48 h. Cells were then washed with pre-warmed PBS and incubated for 1 h with different concentrations of P8-D6 and a respective solvent control  diluted in LCIS (supplemented with 10% FCS). Cells were washed with DPBS and then stained for 20 min with a mixed staining solution containing Hoechst 33258 and CellMaskTM deep red. Subsequently, cells were washed twice with PBS, and clean LCIS was added to the dishes for microscopy. Confocal microscopy was conducted with a Confocal LSM Zeiss 710 equipped with the ELYRA PS.1 system, a Plan-Apochromat 63×/1.4 oil objective (zoom 2×) using three lasers. P8-D6 was detected at 488 nm (green), cell membranes at 647 nm , and nuclei at 405 nm .A total 1 × 10For image analysis, random cells were selected using only the DAPI channel to exclude a biased cell selection. Afterwards, 3D images were reconstructed using the Zeiss Zen software.6 HT-29 cells for 48 h, followed by 1 h incubation with different concentrations of P8-D6 or respective controls. Subsequently, cells were washed with PBS, lysed, and harvested. The lysate was put on a density gradient (0.75–1.6 mg/mL cesium chloride) and centrifuged at 100,000 g for 20 h to separate the heavier Topo–DNA complexes from the lighter free Topos. Fractions of the gradient were blotted on a nitrocellulose membrane, which were after blocking treated with HRP-coupled antibodies to later detect Topo I and both Topo II isoforms using chemoluminescence. Pictures of the membranes were captured with a LAS-4000 imager .ICE assay experiments were carried out as previously described by Subramanian et al. . In brie5 HT-29 cells were seeded in Petri dishes (diameter 3.5 cm). After 48 h growth in complete medium, cells were incubated with different concentrations of P8-D6 and the solvent control (1% (v/v) DMSO) in FCS-free DMEM for 1 h. To gain a positive control, one Petri dish incubated with 1% (v/v) DMSO was subjected to UV-B radiation . Afterwards, all dishes were washed twice with PBS and the cells were harvested using trypsin. Cell viability was checked using trypan blue exclusion. For each sample, an object slide with two agarose pads was prepared, each containing 30,000 embedded cells. After lysis by using a buffer containing Triton X and lauroyl sarcosinate, the slides were washed with PBS and subsequently equilibrated in alkalic electrophoresis buffer (pH 13) for 20 min at 0 °C. Electrophoresis was carried out for 20 min at 25 V, 300 ± 3 mA, 0 °C. Afterwards, slides were neutralized and stained with ethidium bromide (0.02 g/mL in water). Subsequently, microscopy was carried out using a Zeiss Axioskop  and obtained images were analyzed using the “Comet Assay IV” software  to score the tail intensity of 100 nuclei per slide.The comet assay was performed according to the guidelines of Tice et al. , as receTaken together, the results of the present study demonstrated that P8-D6 acts as a dual Topo poison in human colon cancer cells. The observed potency for the stabilization of the DNA–Topo intermediate and for DNA breakage supports the hypothesis that dual Topo poisoning plays a central role in the previously observed potent antineoplastic activity of P8-D6 in vitro, and demonstrates the compound’s high potential for chemotherapeutic use.


N-acetyl-glycoproteins (48 h), while acetate increased (48 h). Less, but different, changes were observed at 4 °C, suggesting different oral and microbial status at different temperatures , and identifying galactose, hypoxanthine, and possibly, choline esters, as potential general stability indicators. After preparation, addition of NaN3 did not impact significantly on saliva stabilization, neither at RT nor at 4 °C, although its absence was accompanied by slight increases in fucose (6.5 h) and proline (8 h) at RT, and in xylose (24 h) at 4 °C. The putative metabolic origins of the above variations are discussed, with basis on the salivary microbiome. In summary, after collection, saliva can be stored at RT/4 °C for up to 6 h and at −20 °C for at least 4 weeks. Upon preparation for NMR analysis, samples are highly stable at 25 °C up to 8 h and at 4 °C up to 48 h, with NaN3 addition preventing possible early changes in fucose, proline (6–8 h), and xylose (24 h) levels.Maintaining a salivary metabolic profile upon sample collection and preparation is determinant in metabolomics. Nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolite changes during short-term storage, at room temperature (RT)/4 °C/−20 °C, and after sample preparation, at RT/4 °C . Interestingly, significant metabolic inter-individual and inter-day variability were noted, probably determining sample stability to some extent. After collection, no changes were noted at −20 °C (at least for 4 weeks). RT storage induced decreases in methylated macromolecules (6 h); lactate (8 h); alanine (12 h); galactose, hypoxanthine, pyruvate (24 h); sarcosine, betaine, choline,  The ss (T1DM) , cardiovs (T1DM) , or cancs (T1DM) . Applieds (T1DM) , to idenN1-acetylspermidine were noted not to change significantly. Up to 8 days at RT, the changes in amino acids were generally larger than in polyamines, but ethanol addition was found to maintain sample integrity. No statistical variations were reported at the remaining temperatures under study [g range) did not impact significantly on saliva composition, as viewed by NMR.Saliva metabolomics has been relatively underexplored, compared to blood or urine, although growing interest has been noted in the last decade ,8, not oer study . Other per study , this ti1H NMR metabolomics study, to our knowledge, of the effects of different storage temperatures and times on saliva composition. This study assesses salivary metabolic changes during storage in conditions, which are particularly relevant upon collection in a typical clinical setting (where −80 °C is not commonly available), and in the laboratory. In particular, we have considered saliva collected for several adult subjects, in the following conditions: (1) immediately after collection at 22 °C and 4 °C  and at −20 °C ; (2) after preparation for NMR analysis, with and without sodium azide , at 25 °C , and at 4 °C .Here, we report the first untargeted 1H NMR spectrum of the saliva of a healthy  adult. The spectrum exhibits a large number of narrow peaks, arising from low molecular weight (Mw) compounds , organic acids, carbohydrates and others, w compounds . Based f saliva ,39,40,41entified , althougentified  [12,38,3N-acetyl-glycoproteins (NAG), urea, and dimethyl sulfone and increased levels of trimethylamine, 5-aminopentanoate, acetate, propionate, leucine, and valine , which became more significant as time evolved towards 48 h  storage of saliva at 22 °C and 4 °C, PCA scores plots a,b showerds 48 h . Lactaterds 48 h . Within rds 48 h , consistrds 48 h . Under rrds 48 h . After tTo evaluate the suitability of a common freezer (at −20 °C) for temporary saliva storage, the profile of samples was investigated up to 48 h (data not shown), having indicated no relevant changes under such conditions. Considering a period of up to 4 weeks, PCA confirmed the large inter-individual difference in salivary profile e, with h3 on salivary metabolic profile was evaluated at 25 °C and 4 °C, over a period of 8 and 48 h, respectively . 3 does not significantly change intra-group dispersion at 25 °C for up to 8 h, conditions that mimic possible standing times prior to acquisition or overnight. Notably, stability seems high already in the absence of NaN3, although intra-group dispersion may be dependent on the individual and day of collection . At 4 °C, but for longer times (up to 48 h)  b, intra-at 25 °C , namely at 25 °C .This study illustrated the significant extension of inter-individual variability in saliva metabolic profile, for healthy donors, as expected, considering the known large diversity of salivary microbiome . One limIn a clinical setting, it is often seldom that a −80 °C freezer is available for ideal immediate sample storage, and a short period of standing time at room temperature or under refrigeration may be required. Notably, saliva did not show statistically relevant changes at either 22 or 4 °C, up to 1 h. However, at 22 °C qualitative changes occur at 3 h, although statistically relevant changes only take place at 6 h, with a decrease in a methyl broad resonance. NMR peak linewidths inversely reflect molecular mobility; hence, we propose that such methyls arise from relatively larger molecules. As the typical NAG methyl resonances at 2.06 ppm only changes (decreases) at 48 h, we suggest that such molecules are not glycoproteins, but possibly other proteins or large molecules. This change may relate to the reported changes in total esterase (TEA) at 6 h at RT , thus puStreptococcus sanguinis, with the release of acetate and ethanol [Veillonella [Streptococcus, Lactobacillus, and Actinomyces spp. [Overall, the larger number of variations at 22 °C appear to express the role of bacterial proteases and saccharolytic pathways, explaining the decrease in pyruvate, galactose, alanine, and choline and the increase in acetate . The dec ethanol ,53, possllonella , whereasces spp. . The decces spp. .The fact that saliva shows delayed degradation post-collection at 4 °C is expected, although a different profile of changes is noted . The mos3 does not seem to be necessary . Still, slight changes were noted in fucose and proline, probably arising from proteolytic cleavage of endogenous glycoproteins, such as glycosylated proline-rich proteins or mucins. Under refrigeration for up to 2 days, saliva also exhibits high stability, again seemingly not requiring NaN3 addition and only exhibiting an increase in xylose (after 24 h), probably also due to glycoproteins hydrolysis. In addition, it is worth recalling that some Gram-positive bacteria, namely Streptococci and Lactobacilli, have been reported to be intrinsically resistant to the oxidant activity of sodium azide [Saliva is here observed to be remarkably stable after preparation, at least up to 8 h at 25 °C, so that adding NaNum azide . Since t2 range. Some of these subjects provided saliva samples in different days, for different parts of the study , and donors were requested to abstain from eating, drinking, smoking, or tooth brushing in the last 1.5 h prior to collection. All collected samples were gently stirred to ensure homogeneity and split into the required number of aliquots. Samples were intentionally not pooled, in order to assess inter-individual variability either in basal salivary profile and/or its stability. For short- (1 h), medium-term (48 h), and post-collection studies (3) were prepared to assess effects of storage at 4 °C  and at 25 °C up to 8 h . In this part of the work, some of the subjects donated saliva in different days, for different assays, hence the discussion of the effects of sodium azide for instance being mainly centered in subjects 1 and 4, for which complete sets of samples were acquired to assess sodium azide and temperature effects.All saliva samples were collected under the approval of the Ethical Committee of the Hospital Center of Coimbra  and signed informed consent forms were obtained from each participating subject. Saliva was collected from 5 healthy female donors (S1 to S5), aged between 21 and 29 years old and body mass indexes in the normal 18.5–21 kg/mhe study . Unstimundations ,61. In o studies a, left,  studies a, right, studies b, two sag, in a Sigma 2-16P centrifuge with r = 82 mm). Then, to 400 μL of the supernatant, 300 μL of sodium phosphate buffer were added: 70 mM Na2HPO4/NaH2PO4, containing 0.1% (m/v) Na+/3-trimethylsilyl-propionate (TSP) and 2 mM NaN3, except in the case of samples aimed at studying the effect of sodium azide absence  with an 8.10–8.97 µs 90 degree pulse, 100 ms mixing time, water suppression (with a power level of 45.57 dB and offset frequency of ca. 2350 Hz) during mixing time and relaxation delay (RD 4 s). The 90-degree pulse length and water suppression conditions were optimized for each sample. A total of 256 transients were acquired into 64 k data points, with spectral width of 10,000 Hz, and 3.28 s acquisition time. Each free induction decay (FID) was multiplied by a 0.3 Hz exponential function prior to Fourier transformation. Spectra were manually phased, and baseline-corrected and chemical shifts were referenced internally to TSP (δ 0.0). Peak assignments were carried out based on literature reports [For each saliva sample, a  reports ,39,40,41 reports , 2D NMR  reports ), and cop-values (Wilcoxon test). Metabolite variations were considered significant if |ES| > ESerror and p-value < 0.05. Spearman correlation analysis was also performed to correlate each metabolite with time length storage for each condition under study. All statistical tests and boxplots were performed using the R-statistical software and MATLAB .After excluding the water region (4.50–5.19 ppm), the spectra were aligned by recursive segment-wise peak alignment  and normWith this work, we aimed at mimicking realistic conditions regarding saliva sampling in the clinic (post-collection) and in the laboratory , in order to propose best practices regarding saliva storage in the short and medium term. While noting a significant inter-individual and inter-day variability in the metabolic profile of saliva post-collection, our results showed that, after collection, saliva may be kept at 22 °C or 4 °C for up to 6 h, after which some metabolite levels start to change . For longer periods, saliva is best stored at −20 °C for at least 4 weeks . We propose that the metabolite changes noted should reflect a distinct interplay of oral and microbial metabolisms at the different temperatures, in tandem with a possible contribution from inter-individual variability. Only galactose, hypoxanthine, and possibly, methylated choline esters were found varying at both 22 °C and 4 °C, suggesting their levels as potential individual-independent indicators of saliva integrity. The stability of saliva after preparation for NMR analysis was evaluated at 25 °C  and at 4 °C . In both conditions, saliva stability was high and, although addition of sodium azide to maintain sample microbiological integrity was found not to have a significant effect on saliva stability, its absence may induce early (6–8 h) increases in fucose, xylose, and proline levels.One limitation of this work relates to the low number of subjects, although multiple collections were made when possible (thus enabling inter-day variability to be assessed). A second important limitation relates to the absence of in tandem oral and microbiological examinations of the donors. Although all subjects regularly attended routine dental appointments, their examination by a calibrated trained dentist would have evaluated important oral status indicators , which would aid in the interpretation of the considerable inter-individual variability found in saliva profile and stability. Nevertheless, we believe that our results help to establish useful guidelines as to how to suitably store saliva samples in a clinical setting immediately post-collection  or after preparation in the lab , thus ensuring sample integrity for NMR metabolomics.


Unresolved inflammation and tissue destruction are considered to underlie the failure of dental pulp repair. As key mediators of the injury response, dental pulp stem cells (DPSCs) play a critical role in pulp tissue repair and regeneration. Resolvin E1 (RvE1), a major dietary omega-3 polyunsaturated fatty-acid metabolite, is effective in resolving inflammation and activating wound healing. However, whether RvE1 facilitates injured pulp-tissue repair and regeneration through timely resolution of inflammation and rapid mobilization of DPSCs is unknown. Therefore, we established a pulp injury model and investigated the effects of RvE1 on DPSC-mediated inflammation resolution and injured pulp repair.A pulp injury model was established using 8-week-old Sprague-Dawley rats. Animals were sacrificed on days 1, 3, 7, 14, 21, and 28 after pulp capping with a collagen sponge immersed in PBS with RvE1 or PBS. Hematoxylin-eosin and Masson’s trichrome staining, immunohistochemistry, and immunohistofluorescence were used to evaluate the prohealing properties of RvE1. hDPSCs were incubated with lipopolysaccharide (LPS) to induce an inflammatory response, and the expression of inflammatory factors after RvE1 application was measured. Effects of RvE1 on hDPSC proliferation, chemotaxis, and odontogenic differentiation were evaluated by CCK-8 assay, transwell assay, alkaline phosphatase (ALP) staining, alizarin red staining, and quantitative PCR, and possible signaling pathways were explored using western blotting.+ and CD105+ DPSCs to the damaged molar pulp mesenchyme. Isolated primary cells exhibited the mesenchymal stem cell immunophenotype and differentiation. RvE1 promoted hDPSC proliferation and chemotaxis. RvE1 significantly attenuated pro-inflammatory cytokine  release and enhanced ALP activity, nodule mineralization, and especially, expression of the odontogenesis-related genes DMP1, DSPP, and BSP in LPS-stimulated DPSCs. RvE1 regulated AKT, ERK, and rS6 phosphorylation in LPS-stimulated DPSCs.In vivo, RvE1 reduced the necrosis rate of damaged pulp and preserved more vital pulps, and promoted injured pulp repair and reparative dentin formation. Further, it enhanced dentin matrix protein 1 and dentin sialoprotein expression and accelerated pulp inflammation resolution by suppressing TNF-α and IL-1β expression. RvE1 enhanced the recruitment of CD146RvE1 promotes pulp inflammation resolution and dentin regeneration and positively influences the proliferation, chemotaxis, and differentiation of LPS-stimulated hDPSCs. This response is, at least partially, dependent on AKT, ERK, and rS6-associated signaling in the inflammatory microenvironment. RvE1 has promising application potential in regenerative endodontics.The online version contains supplementary material available at 10.1186/s13287-021-02141-y. The existence of mesenchymal stem cell populations in adult tissues that conduce tissue cell turnover and respond to tissue damage is, in general, accepted . In dentInflammation resolution has long been considered to be a passive process. However, recent studies have shown that it is an active, programmed process controlled by endogenous specialized pro-resolution lipid mediators (SPMs), including resolvins, lipoxins, protectins, and maresins , 10. TheResolvin E1 (RvE1), one of the SPMs, is of particular interest because of its outstanding anti-inflammatory and pro-resolution effects in various inflammatory disease models. RvE1 has been shown to directly act on tendon stromal cells to promote inflammation resolution in periarticular tendinitis , to acceTherefore, we established a rat pulp injury model to investigate the effects of RvE1 on DPSC-mediated inflammation resolution and injured pulp repair. We observed that RvE1 promoted pulp repair by resolving pulp inflammation, forming reparative dentin, and enhancing the ability of DPSCs to proliferate, chemotax, and differentiate, at least in part by regulating the phosphorylation of AKT, ERK, and ribosomal protein S6 (rS6) in LPS-stimulated DPSCs.All experimental animal studies and the use of human pulp cells were approved by the Institutional Review Board of Tongji University . The experimental procedures abided by ethics standards, and written informed consent was obtained from the volunteers.n = 60) were used [The pulp injury model was established as previously described , 21. Eigere used . The bilere used , 17, 23.The samples were immersed in 4% PFA at 4 °C for 24 h, then demineralized by immersing in a 10% EDTA solution at 4 °C for approximately 2 months, with a solution change every 3 days. The samples were dehydrated using a graded ethanol series, embedded in paraffin, and sectioned at 4-μm thickness. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin  and Masson’s trichrome . We determined the porosity of the dentin bridge by measuring the percentage of the spaces containing cells within the reparative dentin using ImageJ [For immunohistochemical staining, the sections were processed with hyaluronidase and goat serum blocking . Next, they were incubated with primary antibody at 4 °C overnight. The primary antibodies included anti-DMP1 , anti-DSP , anti-ChemR23 , anti-BLT1 , anti-TNF-α , and anti-IL-1β . After washing in PBS, the sections were incubated with biotinylated secondary antibodies and further processed using streptavidin peroxidase and a DAB Detection Kit  per the manufacturer’s instructions. Positively stained areas were measured with ImageJ.For immunofluorescence staining, the sections were subjected to antigen retrieval and treated with goat serum . Then, they were incubated with anti-CD105 , anti-CD146 , anti-ChemR23 , and anti-BLT1  at 4 °C overnight. After washing with PBS, the sections were incubated with fluorescence-labeled secondary antibodies  for 1 h. The sections were counterstained with DAPI and observed under a fluorescence microscope .2. Cell clones were isolated and expanded. Stemness surface markers and pluripotency were evaluated as described previously [Human dental pulp tissue was isolated from normal impacted third mandibular molars or normal premolars for orthodontic therapy from healthy patients (13–20 years old) with informed consent at Tong Ji Stomatology Hospital. We also obtained written parental consent for the minors before the study was begun. Briefly, dental pulp tissue was collected, cut into pieces, and digested with 0.3% collagenase for 30 min to obtain a single-cell suspension. The cells were cultured in minimal essential medium alpha modification  containing 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in 5% COeviously . In brie2. After 14 days, the cells were fixed with 4% PFA, stained with 0.5% crystal violet, washed with distilled water, and dried. Aggregates of 50 cells or more were scored as 1 CFU.Proliferation ability was assessed by a colony-forming unit (CFU) assay. hDPSCs were seeded at 200 cells/well in 6-well plates in growth medium and incubated at 37 °C in 5% COFor the multilineage differentiation assay, alizarin red S, alcian blue, and oil red O staining were used to identify osteogenic, chondrogenic, and adipogenic differentiation, respectively.3 cells/well were seeded in 96-well plates  in α-MEM containing 10% FBS and incubated overnight at 37 °C in 5% CO2. Then, the cells were exposed to 1, 10, 50, 100, or 200 nM RvE1 or control (PBS). After incubation for 1, 3, 5, or 7 days, CCK-8 was added to each well and the plates were further incubated for 2 h. Then, the optical density at 450 nm was read using a microplate reader .For the cell proliferation assay, 2 × 104 cells) in 200 μL of 5% FBS/α-MEM were seeded on the cell culture inserts. In the lower chambers, 750 μL of 5% FBS/α-MEM containing RvE1  or control (PBS) was added. After incubation for 24 h or 48 h, the cells on the upper side of the insert were scraped off a using cotton-tipped swab. The cells that had passed through the insert membrane were fixed with 4% PFA and stained with 0.5% crystal violet. Cells were analyzed in four random fields under an inverted microscope (Nikon Eclipse 80i).The chemotaxis ability of hDPSCs treated with RvE1 or PBS for 24 h or 48 h was assessed in 24-well plates, using Transwell Filter Inserts (Corning). hDPSCs  and supplemented with either 1 μg/mL lipopolysaccharide (LPS), 100 nM RvE1, 1 μg/mL LPS + 100 nM RvE1, or the control (PBS). The medium was changed every 2 days. After 1 and 2 weeks, the medium was removed for ALP staining. The cells were fixed in in 4% PFA for 20 min. After the cells were rinsed with PBS three times, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution  was added to each well for 10 min. Then, the cells were washed and photographed. For quantitative analysis, 10% (w/v) cetylpyridinium chloride solution (Sigma-Aldrich) was added for 30 min, and the staining intensity was quantified by measuring the absorbance at 562 nm in a microplate reader (Bio-Tek).hDPSCs were seeded in 24-well plates (Corning) and cultured in odontoblastic differentiation medium containing 2 mM β-glycerophosphate (Sigma-Aldrich), 50 mg/mL ascorbic acid (Sigma-Aldrich), and 10Mineral deposits in the cultured cells were stained with alizarin red to evaluate odontoblastic differentiation. After 2 and 3 weeks, cells were fixed with 4% PFA and stained with 0.2% alizarin red (Sigma-Aldrich) at room temperature for 20 min. The alizarin red S-positive area was analyzed under a stereoscopic microscope . For quantitative analysis, we used 10% (w/v) cetylpyridinium chloride solution and measured the optical density at 450 nm in a microplate reader (Bio-Tek).TNF-α, IL-1β, and IL-6, for 7, 14, and 21 days to determine mRNA levels of dentin matrix protein1 (DMP1), dentin sialophosphoprotein (DSPP), and bone sialoprotein (BSP). Total RNA was isolated using Trizol reagent. RNA was reverse-transcribed using a Transcriptor First-Stand cDNA Synthesis Kit . qPCRs were run using the FastStart Essential DNA Green Master Kit (Roche) and a LightCycler 96 Instrument (Roche). The details of the protocols were previously described [To evaluate the capability of RvE1 in countering inflammation, hDPSCs were pretreated with LPS (1 μg/mL) to induce an inflammatory condition. Then, 100 nM RvE1 was added and the cells were cultivated for 1, 3, or 7 days to determine mRNA levels of escribed . The pri5) were cultured in 6-well plates in α-MEM for 24 h. Then, the cells were starved overnight in α-MEM plus 1% FBS, gently washed with PBS, exposed to LPS for 30 min, and treated with RvE1 for 15, 30, or 60 min. Thereafter, the hDPSCs were lysed in radio immunoprecipitation buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail . The cells were collected by scraping, lysed, and centrifuged at 8000×g for 20 min. The total protein concentration was measured using a BCA Protein Assay Kit . Proteins (20 μg) were resolved using a 10% PAGE Fast Gel Preparation Kit  and transferred to a nitrocellulose membrane . After blocking in TBST  containing 5% nonfat milk, the membranes were incubated with primary antibodies against pAkt, Akt, pERK1/2, ERK1/2, prS6, rS6  overnight. α-Tubulin  was used as an endogenous control. Next, the membranes were washed thrice with TBST, incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h, and washed thrice with TBST. Then, the membranes were treated with chemiluminescence reagent (Sigma-Aldrich) and visualized by enhanced chemiluminescence. Protein bands were quantified using ImageJ. The assay was performed three times using different samples.hDPSCs  in SPSS 20.0 . In the control group, many samples suffered from serious inflammation and were ending up with necrosis   and dentin sialoprotein (DSP) were labeled as markers of active odontoblasts . At 2 weP < 0.05)  Fig. C.Fig. 3+ and CD105+ cells were mainly detected around the vasculature   in the damaged site are stimulated to proliferate, leave the vessels, and differentiate into odontoblast-like cells that produce a reparative form of dentine to form a dentine bridge . These p05) Fig. B.Fig. 4Primary cells presented a typical homogeneous spindle morphology Fig. A and werA CCK-8 assay was used to evaluate the effect of RvE1 on hDPSC proliferation. Cells were incubated with RvE1  or PBS (control) for 1, 3, 5, or 7 days. On day 1, the proliferation ability unapparent increased after treatment with RvE1, whereas on 3, 5, and 7 days, the proliferation increased markedly after treatment with RvE1 as compared with the control, especially at 100 nM Fig. A. A TranTNF-α, IL-1β, and IL-6) as determined by RT-qPCR  were treated or not with 100 nM RvE1 in the presence of 1 μg/mL LPS for 7 and 14 days for ALP analysis, 14 and 21 days for alizarin red staining, and 7, 14, and 21 days for RT-qPCR analysis.P < 0.05)     group (05) Fig. A. LPS su05) Fig. A. Alizar05) Fig. B. mRNA l05) Fig. C.Fig. 8P < 0.05)  Fig. A, B.FigThe maintenance of vital dental pulp following pulp injury is a matter of debate. Advanced caries or traumatic lesions can irreversibly damage the vital dental pulp to cause an excessive inflammatory response, which is not conducive to repair , 31. TheTNF-α, IL-1β, and IL-6 in LPS-induced hDPSCs. We also showed that RvE1 limited the level of inflammation by decreasing the expression of the typical pro-inflammatory factors TNF-α and IL-1β in rat molar pulp tissue. Therefore, timely resolution of pulp inflammation is particularly important for preserving the vital pulp and reducing the necrosis rate of the damaged pulp. This finding was consistent with a previous finding that RvE1 exerted a protective role against pulp inflammation in infected rat dental tissue [RvE1 promotes inflammation resolution in the pulp microenvironment. In this study, we found that RvE1 decreased the expression of the inflammation-related genes l tissue .We previously reported that RvE1 can suppress the activation of DPFs to inhibit pulp inflammation, at least partially, in a ChemR23-dependent manner . RvE1 reRvE1 not only plays a key role in controlling inflammation, but also stimulates mineralized tissue repair. We found that RvE1 enhanced reparative dentin formation; DMP1 and DSP, which are markers of active odontoblasts that indicate vital pulp tissue integrity and function, were more strongly expressed upon RvE1 application. RvE1 promoted DPSC proliferation, chemotaxis, and differentiation. A previous study revealed an anti-inflammatory role of RvE1 in pulpitis in the short term and suggested that RvE1 may induce the formation of reparative dentine in the longer term, although this was not proven . Our stuRvE1 promoted DPSC commitment in the dental mesenchyme. Pulp mesenchymal stem cells are closely associated with perivascular niches and have commonalities with pericytes . One typWe showed that RvE1 are likely related to the activation of the PI3K-Akt and ERK signaling pathways and the induction of rS6 phosphorylation in LPS-stimulated DPSCs. These results are consistent with those of previous studies on RvE1-ChemR23 signaling in macrophages . Karim eRvE1 reduced the necrosis rate, preserved more vital pulp, and promoted dental pulp repair and regeneration after damage. In vivo, RvE1 application promoted pulp inflammation resolution and induced effective reparative dentin reconstruction in a rat injury model. In vitro, RvE1 enhanced the proliferation, chemotaxis, and odontoblastic differentiation of hDPSCs with or without LPS, which is, at least partially, dependent on AKT, ERK, and rS6-associated signaling in the inflammatory microenvironment. Thus, RvE1 might have potential as a new therapeutic strategy to promote pulp inflammation resolution and tissue regeneration.Additional file 1: Figure S1, Figure S2, Figure S3, Table S1, Table. S2.


Hypoxia reduced aerobic capacity by ∼22% in both groups , while Tibetans consistently outperformed their Han Chinese counterpart by ∼32% in normoxia and hypoxia . We found cerebral [O2Hb] was higher in Tibetans at normoxic maximal effort compared Han (p = 0.001), while muscle [O2Hb] was not different (p = 0.240). Hypoxic exercise lowered muscle [O2Hb] in Tibetans by a greater extent than in Han (interaction effect: p < 0.001 vs. normoxic exercise). Muscle [O2Hb] was lower in Tibetans when compared to Han during hypoxic exercise , but not during normoxic exercise . Muscle [HHb] was not different between the two groups during normoxic and hypoxic exercise (p = 0.778). Compared to Han, our findings revealed a higher brain tissue oxygenation in Tibetans during maximal exercise in normoxia, but lower muscle tissue oxygenation during exercise in hypoxia. This would suggest that the Tibetans privileged oxygenation of the brain at the expense of that of the muscle.The Tibetans’ better aerobic exercise capacity at altitude remains ill-understood. We tested the hypothesis that Tibetans display better muscle and brain tissue oxygenation during exercise in hypoxia. Using near-infrared spectrometry (NIRS) to provide indices of tissue oxygenation, we measured oxy- and deoxy-hemoglobin ([O Tibetans are renowned for their superior exercise performance at high altitude compared to their Western and Han Chinese counterparts . While t2 consumption for a given exercise workload compared to lowlanders, suggesting a better O2 economy , muscle NIRS signals provide unique insights into O2 balance (and therefore oxidative metabolism) in the working muscle  during repeated sprints (Near-infrared spectroscopy (NIRS) provides a non-invasive method of assessing changes in muscle tissue oxygenation during exercise . By deteg muscle . In the  changes . Studies sprints , increme sprints , and sta sprints .2 delivery compared to Han Chinese  and hypobaric hypoxia  in Tibetans and Han Chinese living in Xining  of the Qinghai Province, People’s Republic of China. We tested the hypothesis that when compared to Han Chinese, Tibetans would exhibit higher brain and muscle tissue oxygenation during exercise in hypoxia, but not during exercise in normoxia.Forty male participants were initially recruited into this study. Following saline-contrast echocardiography screening to exclude participants with a patent foramen ovale (see below), twenty male participants completed this study, consisting of ten Han Chinese and ten Tibetans . The TibDeclaration of Helsinki.All the participants were judged healthy by medical history, physical examination, resting electrocardiogram, echocardiogram and respiratory function tests and were not taking any medication. The participants were informed of the experimental procedures and potential risks involved in the study before their written consent was obtained. The study was approved by the University of Oregon Institutional Review Board and the Qinghai High Altitude Medical Science Institutional Committee on Human Research and complied with the 2 gradient  and lower SaO2 during maximal exercise in normoxia . The foramen ovale is an interatrial communication which allows blood flow to bypass the pulmonary circulation during fetal life but normally closes after birth. Importantly, the presence of a PFO in adults has been shown to elevate resting alveolar-arterial Onormoxia . Likewisnormoxia . Therefonormoxia , the pre2 (FIO2) from 0.209 to 0.22–0.24 resulting in a mild increase in inspired O2 .The participants visited the laboratory on three occasions. After a full familiarization with the experimental procedures outlined below (visit one), the participants underwent two experimental exercise sessions (visits two and three) in a pressure-regulated chamber : (i) normobaric normoxia (simulated sea-level by increasing the chamber pressure to 1 atm), and (ii) hypobaric hypoxia , in a randomized, single-blinded, balanced fashion. Due to technical limitations with the chamber, there was a mild increase in fraction of inspired O2 through a face mask, followed by a 3-min washout period to ensure all the variables returned to pre-O2 breathing values; (iii) 4 min resting baseline; (iv) step-incremental cycling until exhaustion; (v) 5 min recovery; and (vi) ramp-incremental cycling until exhaustion. All of the experimental sessions were conducted at the National Key Laboratory of High Altitude Medicine in the city of Xining of the Qinghai Province, People’s Republic of China. Before each experimental session, all participants were asked to abstain from caffeine for 12 h, and alcohol for 24 h.For the normobaric normoxia and hypobaric hypoxia sessions, the experimental protocol was comprised of: (i) 20 min instrumentation; (ii) 4 min hyperoxic exposure, breathing 100% OSeated on a reclining ergometer tilted into a left lateral position , the participants were instructed to begin cycling at 70 watts, at a pedaling rate of 70 rpm. The work rate was increased by 30 watts every 3 min thereafter until the participant reached voluntary exhaustion.–1) thereafter until the participant reached voluntary exhaustion.Following a 5-min recovery period whilst positioned on the ergometer, the participants were instructed to begin cycling at 0 watts, at a pedaling rate of 70 rpm. The work rate was increased by 0.5 watts every second , expired CO2 (V̇CO2), and respiratory exchange ratio (RER) were then calculated by the metabolic cart and expressed in either L.min–1 BTPS (for V̇E) or mL.min–1 STPD (V̇O2 and V̇CO2).Throughout the experimental protocol, the participants wore a facemask attached to a spirometer , from which expired gases and breath-by-breath respiratory flow was monitored using a metabolic cart . Ventilation (V̇E), O2 saturation (SpO2) was measured from the right side of the forehead using pulse oximetry . A three-lead electrocardiogram was used to determine heart rate (HR). Stroke volume (SV) was estimated using transthoracic electrical bioimpedance cardiography . Cardiac output (CO) was subsequently calculated by multiplying SV with HR.Continuous beat-to-beat blood pressure was monitored using finger plethysmography , from which mean blood pressure (BP) was derived from the timed-average of the BP waveform. Peripheral OBilateral middle cerebral artery blood velocities  were measured in the middle cerebral artery using a 2-MHz pulsed Doppler ultrasound system . The Doppler ultrasound probes were positioned over the temporal windows and held in place with an adjustable plastic headband. The MCAv signals were acquired at depths ranging from 43 to 54 mm. Signal quality was optimized, and an M-mode screen shot was recorded to facilitate subsequent probe placements. In our hands, day-to-day reproducibility of MCAv has a coefficient of variation of <10%. The bilateral MCAv were averaged using the following equation to represent global cerebral blood flow (CBF) during rest and exercise:2Hb] and [HHb] concentrations obtained using spatially resolved, continuous wave near-infrared spectroscopy . For the muscle tissue oxygenation, a source-detector spacing of 3.8 cm and a differential pathlength factor of 4.0 were used  calculated using the formula: DPF = 4.99 + 0.067 × age0.814 was measured by monitoring changes in [Oere used . Cerebraage0.814. Total H2Hb], [HHb], and [totHb] are expressed as absolute values (μMol).The cerebral and muscle ) and hematocrit (Hct). Arterial O2 content (CaO2) was subsequently calculated using the equation:Arterial blood gas samples were obtained from a 22-gauge arterial catheter placed into a radial artery; blood samples (2 mL) were taken over approximately five cardiac cycle periods into a pre-heparinized syringe. Following standardized calibration, all blood samples were analyzed using an arterial blood-gas analyzing system  for pH, partial pressure of arterial OAural temperature from the right ear was recorded as a surrogate of core body temperature. The blood gas values were temperature corrected .Energy expended (EE) was calculated using the equation :–1, V̇O2 is O2 uptake in L.min–1 and V̇CO2 is the CO2 expired in L.min–1.where, EE is energy expended in J.sGross efficiency (GE) during steady-state exercise was calculated using the equation :–1.Where GE is gross efficiency in percentage, work rate is in watts, and EE is energy expended in J.s2 uptake and expressed as kJ.L–1.Exercise economy (EC) was calculated as power output divided by O2, V̇CO2 and RER which were recorded on the metabolic cart, all analog data were sampled and recorded at 200 Hz on a computer for off-line analysis .Except for V̇E, V̇Ot test with Welch’s correction was used to compare the participants’ characteristics between Han Chinese and Tibetan . Analyses of resting parameters were performed on averaged data from the last 2 min of the baseline period, and from the last 1 min of the hyperoxic exposure and recovery period. During exercise, the mean values during the last 30 s of workload , and the mean value of the last 30 s of the ramp incremental exercise for maximal exercise effort (MAX) were extracted for analysis.Unpaired 2 breathing, at rest, during steady-state exercise , recovery and maximal exercise effort were assessed using mixed linear model analysis . For significant effects and interaction between hypoxia effect and group effect, post hoc tests were performed using Sidak’s adjustment for multiple comparisons (α-level of 0.05). In addition to p-values, Cohen’s d values (effect size) are reported for altitude and group effects. Cohen’s d value was calculated using the formula  and group (Han Chinese vs. Tibetans) on arterial blood gases, cardiorespiratory variables, cerebral haemodynamics and muscle tissue oxygenation during 100% O formula :1 and M2 are means of group 1 and 2; σpooled is the standard deviation of the pooled data. The effect sizes were classified as (d < 0.2); small (d ≥ 0.2); medium (d ≥ 0.5); large (d ≥ 0.8); and very large (d ≥ 1.3). Data are reported as mean ± SD in text, tables and figures.where, Mified as : negligi2 breathing increased muscle [O2Hb] by ∼2.9 μM in both groups , while neither muscle [HHb]  nor [totHb] were affected . There was a tendency for muscle [HHb] to be lower by ∼2.1 μM in Tibetans during 100% O2 breathing , while no significant group effects were observed (p > 0.05).In hypoxic conditions, 100% OF = 31.5, d = 0.8, p < 0.001, F = 8.8, d = 1.0, p = 0.008).Hypoxia reduced aerobic capacity by ∼22% in both Han Chinese and Tibetans compared to their normoxic values (2 by ∼85.6 mmHg (F = 1778.1), PaCO2 by ∼6.3 mmHg (F = 112.7), SaO2 by ∼16.8% (F = 132.8) and CaO2 by ∼3.0 mL O2.dl–1 (F = 10.51), and elevated resting pH by ∼0.05 (F = 60.5) in both Han Chinese and Tibetans , PaCO2 by ∼13.7 mmHg , SaO2 by ∼17.5% , CaO2 by ∼3.2 mL O2.dl–1  in both Han Chinese and Tibetans, while it elevated [Hb] by ∼0.8 g.L–1  and pH by ∼0.09 , PaCO2 by ∼16.7 mmHg , SaO2 by ∼16.6% , CaO2 by ∼2.9 mL O2.dl–1 in both groups , while pH was elevated by ∼0.10 compared to normoxia , CO by ∼1.0 L.min–1  and lowered SpO2 by ∼17.7%  compared to normoxia , particularly in hypoxia , but was not different in normoxia (p = 0.917). Hypoxia selectively lowered resting RER in Han Chinese . As a result, hypoxia lowered resting RER in Han Chinese by ∼0.054 , while no change was observed in Tibetans .In both Han Chinese and Tibetans, exposure to hypoxia elevated resting HR by ∼19.6 b.min–1  and lowered SpO2 by ∼20.1% , V̇O2 by ∼283 ml.min–1  and V̇CO2 by ∼251 ml.min–1 , but had no effects on CO or BP . There was a group difference on RER during step-incremental cycling , which was mediated by a non-significant group difference in V̇CO2 . In both normoxia and hypoxia, RER was lower in Tibetans by ∼0.097 (d = 1.1) throughout the step-incremental cycling compared to Han Chinese.For a given workload, hypoxia elevated HR by ∼10.2 b.min2 by ∼11.9%  compared to normoxia, but had no effects on HR  or BP .During recovery, hypoxia lowered SpO2 by ∼15.6% , V̇O2 by ∼462 ml.min–1 ), V̇CO2 by ∼462 ml.min–1  and CO by ∼1.7 L.min–1 , but had no effect on HR or BP .During maximal effort, hypoxia lowered SpOp < 0.05, –1 . Irrespective of the condition, GE was higher by ∼5%, in Tibetans at maximal effort compared to Han Chinese , while EC was higher by ∼0.18 kJ.L–1 , but had no effects on muscle [totHb]  or [O2Hb] . No group effects were observed in resting muscle NIRS signals , and elevated muscle [HHb] by ∼4.7 μM , while muscle [totHb] was unchanged . Hypoxia lowered muscle [O2Hb] by ∼7.6 μM in Tibetans  but not in Han Chinese , but not during normoxic exercise . Post hoc analysis showed that hypoxia selectively increased muscle [totHb] in Han Chinese , but not in Tibetans . We found no significant group effects on muscle [HHb] during step-incremental cycling .We observed an interaction between hypoxia and group on muscle [totHb]  and [totHb] by ∼3.5 μM , but had no effect on resting MCAv  or cerebral [O2Hb] , and lowered cerebral [O2Hb] by ∼7.9 μM  and cerebral [totHb] by ∼4.1 μM , this reduction was greater in the Han Chinese group , while hypoxia only tended to lower MCAv in Tibetans by ∼4.1 cm.s–1 . Tibetans had lower MCAv compared to Han Chinese in normoxia , while they were not significantly different in hypoxia , [HHb]  or [totHb] during step-incremental cycling  and cerebral [totHb] by ∼4.3 μM , compared to normoxia but not MCAv  or cerebral [HHb] , cerebral [O2Hb] by ∼10.9 μM  and cerebral [totHb] by ∼6.9 μM , and elevated cerebral [HHb] by ∼4.1 μM , but only in normoxia . Compared to Han Chinese, cerebral [O2Hb] was higher in Tibetans at maximal effort in normoxia by ∼8.9 μM , but not in hypoxia . As a result, hypoxia reduced cerebral [totHb] during maximal effort in Tibetans by ∼10.8 μM , but not in Han Chinese .At maximal effort, hypoxia lowered MCAv by ∼13.8 cm.s2Hb] and [totHb] at maximal effort compared to Han Chinese; (3) In hypoxia, Tibetans displayed lower muscle [O2Hb] compared to the Han Chinese, which was not mediated by any differences in SaO2 or CaO2; and (4) For a given workload, Tibetans exhibited greater muscle desaturation during exercise in hypoxia, but not in normoxia. It follows that during exercise in hypoxia Tibetans as compared to Han Chinese seem to defend their brain oxygenation over muscle oxygenation without any obvious cost to performance.The underlying mechanisms responsible for Tibetans’ superior work capacity at altitude remains unclear. We compared cerebral and muscle tissue oxygenation responses to step-incremental cycling and at maximal exercise during ramp incremental cycling between Tibetans and Han Chinese in normobaric normoxia and hypobaric hypoxia in a pressure-regulated chamber, simulating altitudes equivalent to sea-level and 5,000 m altitude, respectively. Our main findings are that: (1) Irrespective of the condition, Tibetans consistently outperformed their Han Chinese counterparts and exhibited better economy at maximal exertion; (2) In normoxia, Tibetans displayed lower MCAv during submaximal exercise, yet achieved higher cerebral [O2Hb] between exercise at low altitude and at 4,300 m in lowlanders, while muscle [HHb] was increased at high altitude, suggesting greater O2 extraction. Hypoxic training has been shown to enhance muscle [HHb] response to hypoxic exercise, while muscle [O2Hb] response was unaffected , but lower muscle perfusion (less [O2Hb] and [totHb]). Our muscle NIRS data allude to lower muscle O2 utilization in Tibetans for a given workload in hypoxia compared to Han Chinese. Furthermore, Tibetans achieved higher maximal workload despite similar V̇O2max in normoxia, and thus exhibited better exercise economy  Han Chinese at 4,700 m, along with greater maximal work at a lower V̇O2max. These findings allude to an enhanced muscle O2 economy, presumably due to a metabolic adaptation in Tibetans. Intriguingly, this enhanced muscle O2 economy appears to persist following descent to lower altitude  compared to fatty acid oxidation (4.1 unit.mol O2–1)  . Therefoe muscle . There ie muscle . The cape muscle . Additioe muscle . PPARα ee muscle . At reste muscle . As revi2 in Tibetans. In altitude-born Sherpas, 2. Since these muscle characteristics were similar between Sherpas and acclimatized Caucasian climbers, it is likely the result of adaptation to extreme altitude per se, rather than a unique feature of the Himalayan natives. In addition, lower mitochondrial densities have been observed in both high altitude and lowland-dwelling Sherpas and Tibetans, resulting in a higher maximal O2 consumption-to-mitochondrial volume ratio  and HR (∼13 b/min) in Tibetans during treadmill running in hypoxia compared to their Han Chinese counterparts  and postural position (reclined and tilted vs. upright).We previously found higher SpOterparts . Furtherexercise . Given t2 values we obtained in normoxic conditions were higher than expected  Second, muscle oximetry based on NIRS signals provides non-invasive and region-specific information of changes in  response to hypoxic exercise nor cerebral NIRS parameters during normoxic exercise, despite a ∼50% improvement in maximal workload (+22 ml.min–1.kg V̇O2max) in their participants. Recently, 2 availability and muscle O2 extraction with improved fitness. Meanwhile, we found comparable muscle tissue oxygenation responses between Han Chinese and Tibetans during incremental exercise in normoxia, and lower muscle O2Hb and totHb in Tibetans during hypoxic exercise. We contend that the between-group differences in muscle tissue oxygenation during incremental exercise is due to ethnicity rather than fitness.Finally, we recruited a small sample  enabled them to maintain a higher cerebral tissue oxygenation at maximal effort compared to Han Chinese. During hypoxic exercise, we found evidence of greater muscle tissue deoxygenation in Tibetans for a given workload, which we interpret as enhanced muscle OThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of Oregon Institutional Review Board and the Qinghai High Altitude Medical Science Institutional Committee. The patients/participants provided their written informed consent to participate in this study.J-LF, TW, AL, and BK contributed to the conception and design of the study. J-LF, TW, LN, WLB, AL, and BK performed the data collection. J-LF, AL, and BK carried out the analysis, carried out the interpretation of the data, and contributed to the revision of the manuscript. J-LF drafted the manuscript and prepared the figures. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.