Usnea baileyi, Usnea aciculifera, Parmotrema dilatatum, and Parmotrema tsavoense were tested against dengue virus serotype 2. Two depsides and one depsidone inhibited dengue virus serotype 2 without any apparent cytotoxicity. Diffractaic acid, barbatic acid, and Parmosidone C were three active compounds further characterized for their efficacies (EC50), cytotoxicities (CC50), and selectivity index . Their EC50 (SI) values were 2.43 ± 0.19 (20.59), 0.91 ± 0.15 (13.33), and 17.42 ± 3.21 (8.95) μM, respectively. Diffractaic acid showed the highest selectivity index, and similar efficacies were also found in dengue serotypes 1–4, Zika, and chikungunya viruses. Cell-based studies revealed that the target was mainly in the late stage with replication and the formation of infectious particles. This report highlights that a lichen-derived diffractaic acid could become a mosquito-borne antiviral lead as its selectivity indices ranged from 8.07 to 20.59 with a proposed target at viral replication.Dengue is a mosquito-borne flavivirus that causes 21,000 deaths annually. Depsides and depsidones of lichens have previously been reported to be antimicrobials. In this study, our objective was to identify lichen-derived depsides and depsidones as dengue virus inhibitors. The 18 depsides and depsidones of  Flaviviridae consisting of a 10 kb single-stranded RNA genome enclosed in an ER-derived envelope. Transmission is caused by a mosquito bite, and the virus infects local dermal cells, including dendritic cells or antigen-presenting cells. Infected dendritic cells travel to the lymphatic and blood circulation and then infect remote organs, causing systemic infection. Secondary heterotypic infection or secondary infection with a different DENV serotype contributes to pathologically robust but incompetent immunity with excessive proinflammatory cytokines. The primary treatment is still supportive, and no specific drug is currently available. The only commercial vaccine is dengvaxia, which the CDC recommended for 9–16 years old with a laboratory-confirmed previous DENV infection and living in an endemic area. Drug candidates such as ivermectin and celgosivir failed the proof-of-concept antiviral efficacies in clinical trials as their effective dosage exceeded the pharmacokinetic dosage [Dengue infection has become a global burden that causes an estimated 100 million cases and 21,000 deaths annually, according to the World Health Organization (WHO) 2022. Clinical manifestations include high fever, myalgia, thrombocytopenia, plasma leakage, hemorrhage, hypovolemic shock, and multiple organ failure. The causative agent is dengue virus consisting of four serotypes (DENV1–4). Evidence suggests that DENV2 is likely correlated to severity [c dosage .Flaviviridae, these compounds could potentially inhibit DENV. In this study, we screened 18 depsides and depsidones extracted from lichens and reported that diffractaic acid was the most potent inhibitor of DENV, in addition to inhibiting other mosquito-borne viruses.Lichens are known for their medicinal properties across cultures, especially in temperate and arctic climates ,5. The f1a) and barbatic acid (1b), showed DENV2 inhibition at 99.98% ± 0.04% and 99.99% ± 0.02%, respectively, without apparent cytotoxicity (2a), galbinic acid (3a), and norstictic acid (3b), were less potent at 96.48% ± 2.90%, 80.50% ± 4.85%, and 78.00% ± 7.55%, respectively. All compounds did not show apparent toxicity under the tested conditions (10 µM compound in 1% DMSO), suggesting that depsides and depsidones were not toxic to Vero cells  with at least 90% inhibition were then further analyzed for efficacies (EC50) and cytotoxicities (CC50). The results showed that diffractaic acid (1a) had the highest selectivity index of 20.59 with an EC50 of 2.43 ± 0.19 µM (50 of 50.13 ± 7.45 µM (1b) was more effective than diffractaic acid with a lower EC50 at 0.91 ± 0.15 µM (50 of 12.10 ± 0.38 µM (2a) was the least effective of the three compounds with the EC50 at 17.42 ± 3.21 µM  strain in Vero cells with a compound concentration of 10 µM. Two depsides, diffractaic acid  varied between approximately 40 and 70 µM with the lowest and highest CC50 in HepG2 and HEK-293 cells, respectively  (50 of 6.21 ± 0.69 µM) (50 of 19.93 ± 1.94 µM) , Zika virus (ZIKV), and chikungunya virus (CHIKV) were carried out in Vero cells , while t4.90 µM) . However0.69 µM)  and poor1.94 µM) . TherefoThe molecular target of diffractaic acid was screened using a time-of-addition assay with low viral titer to initially determine whether the target resides at early or late timepoints. Diffractaic acid at 10 µM was added to DENV2-infected Vero cells (MOI of 0.1) at various timepoints  A. DMSO wTo further determine the dynamic effect of diffractaic acid on a single round of DENV2 infection, time-of-addition (TOA) and time-of-removal (TOR) assays were established A,B. BrieFurthermore, the viral genome levels of the compound-treated group were reported as a proportion of the corresponding DMSO controls D. The re2+-binding sites of the ATPase/RTPase viral enzyme. We conclude that this is likely an accidental finding.Additionally, the DENV2 16,681 was continuously subpassage under the selective pressure of a sublethal dose of diffractaic acid and DMSO control  before a50) of 49.30 ± 1.86 µM  with IC50 over 100 µM [50 result, the depsidone core is unlikely the active candidate for further analysis.Depsides and depsidones are lichen-derived secondary metabolites with several biological activities such as anticancer, antibacterial, antioxidant, antiparasitic, and antiviral ,15,16,17r 100 µM . However50 = 12.10 ± 0.38 µM), corresponding to previous reports [50s of 39.32 to >100 µM in HepG2, HEK-293, THP-1, Huh-7m and RD cells. The toxicity was higher in cancer cell lines than normal cell lines . Our finding corresponds to previous reports on the cytotoxic activity to cancer cell lines and antitumor activity [50) was 962 mg/kg orally [50s. Therefore, diffractaic acid could be less toxic toward animals than observed in the cell-based system.The selectivity index revealed that diffractaic acid was the best among the three compounds (SI = 20.59), being twofold higher than that of barbatic acid. Moreover, a previous report suggested that barbatic acid was cytotoxic to many cancer cells but not to PBMCs ,13. Our activity ,25,26. Fg orally . The let50, was slightly less effective against an enveloped mosquito-borne alphavirus (CHIKV), and was not effective against a non-enveloped enterovirus (EV-A71). However, enterovirus replication is distinct from that seen for DENV, ZIKV, and CHIKV, especially in terms of IRES-dependent translation and the requirement of primers and host-derived factors for replication [In addition, diffractaic acid broadly inhibited DENV1–4 and ZIKV with similar EClication . TherefoFlaviviridae, at the entry and replication stage with an efficacy of 11.8–22.3 µM [50 of diffractaic acid against dengue methyltransferase was more than 20 times higher than the EC50 in the cell-based assay. Therefore, it is possible that methyltransferase is only a contributing factor. Thus, the major target remains to be explored.Time-course experiments and the functional assays revealed that diffractaic acid inhibited DENV virion progeny after entry by interfering with viral replication. The compound could bind reversibly to the target, and viral RNA was the most sensitive parameter. Another lichen-derived depside, atranorin and its derivatives, also potently inhibited the hepatitis C virus, a member of the family –22.3 µM . The comUsnea aciculifera [Usnea baileyi [Parmotrema dilatatum [Parmotrema tsavoense [1H, 13C, and 2D NMR methods as described previously. Briefly, air-dried parts of U. aciculifera (2.02 kg) were ground using the maceration method at room temperature and extracted with n-hexane, dichloromethane , ethyl acetate (EtOAc), and methanol (MeOH) (4 × 10 L). The filtered solution was evaporated under reduced pressure to produce the n-hexane extract (50.0 g), DCM extract (101.0 g), EtOAc extract (110.0 g), and MeOH extract (55.0 g), respectively. The DCM extract (101.0 g) was separated by quick column chromatography using gradient elution with n-hexane–EtOAc (stepwise 80:20–0:10) and EtOAc–MeOH (stepwise 10:0–0:10) to give 13 fractions, DCM.1–DCM.13. DCM.1 (28.0 g) was subjected to a silica gel column eluted with n-hexane–EtOAc (85:15) to give seven subfractions 1.1–1.7. Subfraction 1.1 (4.95 g) was chromatographed with a Sephadex LH-20 column (100 g) with MeOH–DCM (60:40), followed by chromatography with normal silica gel eluted with hexane–DCM (60:40) to produce methyl orselinate  and 7-hydroxy-5-methoxy-6-methylphthalide . Subfraction 1.2 (1.8 g) was chromatographed with the Sephadex LH-20 column (100 g) with MeOH–DCM (60:40) followed by chromatography with RP-C18 silica gel eluted with H2O–MeOH (60:40) to give norstictic acid . Subfraction 1.3 (7.9 g) was subjected to a silica gel column using gradient elution with n-hexane–EtOAc (85:15–0:100) to give barbatic acid  and diffractaic acid .The two depsides and 14 depsidones were extracted from culifera , Usnea b baileyi , Parmotrilatatum , and Parsavoense  , as well as comparison of their NMR data with those of the literature  at a final concentration of 50 mM and stored in an aliquot at −20 °C until use.®CCL-81), LLC/MK2 (ATCC®CCL-7), C6/36 (ATCC®CRL-1660), HepG2 (ATCC®HB-8065), HEK-293 (ATCC®CRL-1573), and THP-1 (ATCC®TIB-202) cells were maintained as previously described [®CRL-1573) were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C with 5% CO2.Vero (ATCCescribed . Huh-7 cescribed . RD cell2.Reference strains of DENV1 (16007), DENV2 , DENV2 (16681), DENV3 (16562), DENV4 (c0036), ZIKV (SV0127/14), and CHIKV (ECSA genotype) were propagated in C6/36 cells as previously described . EV-A71 4) were seeded in a 24-well plate and incubated at 37 °C under 5% CO2 overnight. Cells were infected with DENV2 NGC at a multiplicity of infection (MOI) of 0.1, and compounds were added at 10 µM; DMSO at 1% was used as a no inhibition control. Cultures were incubated for 1 h with gentle rocking every 15 min. Cells were washed with PBS, and cultured in MEM supplemented with 1% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin; 10 mM HEPES was added in the presence of the compound at 10 µM. Cells were incubated at 37 °C with 5% CO2 for 3 days. The supernatants were collected to determine the viral titers using a plaque titration assay as previously described [Vero cells (5 × 10escribed . Compoun50). Each compound was serially diluted to 10 different concentrations and added to DENV2-infected Vero cells (MOI of 0.1), unless otherwise indicated. Cells were incubated for 3 days, and supernatants were collected for plaque titration assay. The efficacy of each compound was calculated from the fit of the nonlinear regression curve and the concentration required for the reduction of 50% viral titer (EC50) was determined. Results were reported as the mean and the standard error of mean (SEM) from three independent experiments.The selected compounds were further analyzed for the effective concentration (EC4) were seeded in a 24-well plate and incubated as described. Cells were infected with DENV2 NGC at an MOI of 0.1 for 1 h with gentle rocking every 15 min. Diffractaic acid at 10 µM was added at various timepoints: 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, and 72 h post infection (hpi). DMSO at 1% was a mock treatment or no inhibition control. After 72 h of incubation, supernatants were collected to determine viral titers using a plaque titration assay. The results were confirmed by three independent experiments.The possible target of the compound was initially screened by a time-of-addition assay . Vero ceTo further investigate the role of the compound in the interference viral replication cycle, we performed the time-of-addition and time-of-removal assays. Vero cells were infected with DENV2 NGC at an MOI of 1 for 1 h with gentle rocking every 15 min. For the time-of-removal assay, the 10 µM diffractaic acid was added to DENV2-infected cells after infection. The compound was removed at the indicated timepoints after infection by washing with PBS and replacing with maintenance medium. For the time-of-addition assay, 10 µM diffractaic acid was added at the indicated timepoints after infection. DMSO at 1% was used as a mock treatment. After 48 h of incubation, the supernatants were collected to determine viral titers, viral RNA, and viral protein using the plaque titration assay, RT-qPCR, and Western blot, respectively. The results were confirmed by three independent experiments.4) were seeded in a 96-well plate and incubated at 37 °C under 5% CO2 overnight. Compounds were added and incubated for 2 days. DMSO at 1% was used as a mock treatment. Cytotoxicity was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit  according to the manufacturer’s instructions and analyzed by spectrophotometry at 450 nm.AThe cytotoxicity of the compounds was also accessed at 10 µM in parallel with viral inhibition screening. Vero cells (1 × 1050) of effective compounds was analyzed by diluting to 8–10 concentrations and adding them to the Vero cells, unless otherwise indicated. Cultures were incubated for 2 days. Cytotoxicity was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) kit  according to the manufacturer’s instructions and analyzed by spectrophotometry at 450 nm.A The cytotoxicity of each compound was calculated from the nonlinear regression curve, and the concentration required for 50% cell death (CC50) was determined. Results were reported as the mean and standard error of mean (SEM) from three independent experiments.The cytotoxic concentration (CC4) were seeded in a 24-well plate and incubated as described. DENV2 NGC (MOI of 0.1) was inoculated into the cells at 4 °C for 1 h. Then, 10 µM diffractaic acid was incubated with DENV2 prior to addition to cells (pre-incubation), co-incubated with DENV2 (co-incubation), or added to DENV2-infected cells after 1 h of incubation (post-attachment incubation). Cultures were maintained for 2 days, and supernatants were collected to determine viral titers by plaque titration assay. The results were confirmed by three independent experiments.Vero cells (5 × 105) were seeded in a 24-well plate and incubated at 28 °C overnight [C6/36 cells  with a Step-OnePlus Real-Time PCR System ABI 7500  as previously described [Viral RNA in infected cells was extracted using NucleoZOL™ reagent  and a Direct-zol™ RNA MiniPrep Kit  according to the manufacturer’s instructions. The viral genome in the experiment was determined by quantitative RT-PCR. The RT-qPCR was performed using a Power SYBRescribed . The β-aescribed .g for 20 min to obtain the cell lysate. For DENV2 protein detection, cell lysates were subjected to SDS-PAGE under nonreducing conditions. The 4G2 monoclonal antibody  was used as the primary antibody for the DENV2 E protein. Mouse anti-β-actin antibody  was used as a loading control. HRP-conjugated goat anti-mouse IgG antibody was used as a secondary antibody . The chemiluminescence signals were developed using Immobilon Classico Western HRP substrate . Protein band intensities were quantified using ImageJ software version 1.53.The infected cells were lysed using NP-40 lysis buffer ) and centrifuged at 12,000× The crystal structures of DENV viral proteins (NS2/3 protease (2FOM ), NS3 he2, 0.4 mg/mL BSA, and 4 mM DTT)) was mixed with 5 µL of various concentrations of diffractaic acid and incubated at room temperature for 10 min. Subsequently, 10 µL of 2.5 µM S-adenosylmethionine (SAM) was added and incubated at 37 °C for 10 min. After incubating, 25 µL of MTase-Glo detection solution was added and incubated at room temperature for 60 min. The luminescence was measured using a VICTORTM X3 2030 Multilabel Reader . The percentage enzyme activity was calculated from two independent experiments using PRISM version 9 .The MTase assay for compound inhibition testing was adapted and optimized as previously described . Each reThe antiviral activities of a lichen metabolite, diffractaic acid, against DENV1–4 and ZIKV, were reported for the first time. The cell-based studies and molecular modeling suggested the possible targets of the virus and the host components involved in viral replication, likely the viral methyltransferase and other targets. Further studies are required to identify the target molecules.


Five-fluorouracil, folinic acid, oxaliplatin and irinotecan (FOLFOXIRI) regimen is used as the first-line treatment for metastatic colorectal cancer (mCRC). The use of capecitabine, an oral fluoropyrimidine pro-drug, is feasible and safe; hence, it provides an interesting alternative to 5-fluorouracil in the abovementioned regimen. This study aimed to evaluate the efficacy and safety of capecitabine, oxaliplatin, and irinotecan (XELOXIRI) regimen use with or without targeted drugs in Chinese patients with mCRC.We conducted a retrospective, longitudinal cohort study of patients with mCRC who received XELOXIRI regimen with or without targeted drugs (bevacizumab or cetuximab) every 2 weeks between January 2017 and November 2019 at the National Cancer Center/Cancer Hospital, the Chinese Academy of Medical Sciences, and Peking Union Medical College. Treatment efficacy was assessed by investigators by evaluating the objective response rate (ORR) and disease control rate (DCR). Overall survival (OS) was assessed using Cox proportional hazards models. The adverse events were also analyzed.RAS/BRAF wild-type  and metastatic organs > 2  were independent prognostic factors for OS. The incidence of any grade of adverse events (AEs) was 96.7% (59/61). Grade ≥ 3 AEs included neutropenia (19.7%), leukopenia (9.8%), diarrhea (3.3%), vomiting (3.3%), febrile neutropenia (1.6%), and thrombocytopenia (1.6%). No treatment-related death occurred.Sixty-one consecutive patients were examined and followed up for survival. As of November 8, 2021, the median follow-up time was 35.4 months. Disease progression and death occurred in 50 (82%) and 38 (62%) patients, respectively. The median treatment duration of XELOXIRI with or without bevacizumab or cetuximab was 10 cycles . The median OS and PFS were 32.2 months (95%CI [24.8–39.6]) and 9.3 months (95% CI [8.1–10.5]), respectively. The ORR of 48 patients with measurable lesions was 70.8%, and the DCR was 89.6%. The use of the XELOXIRI regimen with or without a targeted drug was effective, with a manageable toxicity profile in Chinese patients with mCRC.The online version contains supplementary material available at 10.1186/s12885-022-09889-3. Moreover, indirect comparisons have shown a higher incidence of neutropenia in Asian patients than in Western patients ), and the 1-, 2-, and 3-year OS rates were 84.6%, 55.1%, and 38.6%, respectively. The median PFS was 9.3 months (95% CI [8.1–10.5]), and the 1-year PFS rate was 35.0%. Regarding tumour response, assessed by investigators, none of the 48 patients with measurable lesions achieved CR, whereas 34 achieved PR, resulting in an objective response rate of 70.8% (95% CI [64.1%-77.5%]). The DCR was 89.6% (95% CI [82.9%-96.3%])   . Baseline neutrophil/lymphocyte ratio (NLR) values were available in 52 patients. The median NLR was 2.98 (0.87–31.81); moreover, no significant differences in PFS  and OS  were found between patients with NLR ≥ 3 and NLR < 3.Regarding genetic mutation status, there were significant differences in OS between patients with the 95% CI 0.5–0.91; pRAS/BRAF WT  and more than two metastatic organs  were independent prognostic factors for OS . The main reasons for discontinuing XELOXIRI therapy were surgery (42.6%), switching to maintenance therapy (capecitabine with or without bevacizumab) (14.8%), drug toxicity (6.6%), disease progression (19.7%), loss to follow-up (8.2%), receiving other location therapies (ablation [1.64%] and radiotherapy [4.92%]).2/week and 37.31 mg/m2/week, respectively. Both the mean and median dose intensities of irinotecan were 71.41 mg/m2/week. The mean and median dose intensities of capecitabine were 5599.75 mg/m2/week and 5599.75 mg/m2/week, respectively  underwent drug discontinuation, of whom 10 (16.4%), 5 (8.2%), 1 (1.6%), 2 (3.3%), and 1 (1.6%) patients discontinued irinotecan, oxaliplatin, capecitabine, both oxaliplatin and irinotecan, and both irinotecan and capecitabine, respectively. The mean and median dose intensities of oxaliplatin in the combined treatment regimen were 36.95 mg/m1.2% undeTable This retrospective study showed that the use of the triplet XELOXIRI combination with or without an antibody (bevacizumab or cetuximab) as first-line therapy in Chinese patients with mCRC produces a comparable safety and efficacy to that of the FOLFOXIRI regimen.BRAFV600E mutant in our cohort was higher (19%) than that in the GONO trials (4.8%-10%)2, 3, 5–7. Moreover, 23% of patients in the present cohort had peritoneal metastases; mCRC was most difficult to treat in these patients.The median OS of our cohort was 32.2 months, which is higher than that reported in serial phase III trials conducted by the GONO group (22.6–29.8 months) –6. NotabBRAFV600E mutation is related to the poor prognosis of patients with colorectal cancer, which is non-responsive to anti-EGFR treatment [V600E mutation [BRAFV600E mutation, which motivated the recommendation of the FOLFOXIRI-bevacizumab combination use by international guidelines. However, in the post-hoc subgroup analyses of the TRIBE2 trial based on the BRAFV600Emutational status, no significant differences in efficacy were detected between the use of FOLFOXIRI plus bevacizumab and that of the two-drug chemotherapy plus bevacizumab [BRAFV600E mutation received XELOXIRI plus bevacizumab therapy; the ORR was 50%, and the mOS of 28 months seemed more promising than that reported in the previous trials3, 6 , including clinical characteristics, pathological features, and molecular alterations [BRAF mutations, including subtypes based on signaling mechanisms  [BRAFV600E (BRAFV600E mutant 1 and BRAFV600E mutant 2) [According to a previous study, patients with mCRC having left-sided primary tumours have a better prognosis than those with right-sided primary tumours . Approxirognosis .Several erations –44. At pand III) , moleculand III) , and theutant 2) . NeverthRecent international guidelines have recommended the use of NED to evaluate therapeutic efficacy in patients with colorectal liver-limited metastases , 48–55. NLR has been reported as a poor prognostic factor in several gastrointestinal tumours –59. A reSo far, XELOXIRI administered every 2 weeks with or without bevacizumab has been investigated on patients with different dose schedules in three phase II and one phase I trials conducted in Western countries; the efficacy was found to be promising , 28, 60;2), oxaliplatin (85 mg/m2), and 1–7 or 2–6 days of capecitabine (2000 mg/m2/day) every 2 weeks. The incidences of grade 3/4 neutropenia and diarrhea ranged from 6%-30% and 24%-31%, respectively [2) might result in a lower incidence of grade ≥ 3 diarrhea (11%) and a comparable incidence of grade ≥ 3 neutropenia [2, oxaliplatin 85 mg/m2, and 1–7 days of capecitabine 2000 mg/m2/day) every 2 weeks was a phase I trial conducted on 6 Japanese patients in the neoadjuvant setting. Grade 3/4 neutropenia occurred in 2/6 (33.3%) patients [Italian regimens composed of irinotecan .The use of the XELOXIRI regimen with or without a targeted drug was effective, with a manageable toxicity profile in Chinese patients with mCRC.Additional file 1: Supplementary Table 1. Patient survival. Supplementary Fig. 1. Cox metanalysis of the impact of research factors on survival or risk rate.


It was found that Cu/SiO2-HT-8.5 catalyst prepared by hydrothermal method showed excellent activity in HMF hydrohydrolysis reaction. Under the optimal reaction condition, the total yield of liquid fuels reaches 91.6% with 57.1% yield of DMF and 34.5% yield of DMTHF in THF solvent. Characterizations such as XRD, H2-TPR, N2-adsorption/desorption, TEM and XPS revealed that the Cu particles in the Cu/SiO2-HT-8.5 catalyst have uniform size and high dispersion. The Cu species and the SiO2 support have relatively weak interaction and are easy to be reduced to Cu0, which makes it show excellent activity in the hydrogenolysis of HMF.Efficient conversion of biomass-derived 5-hydroxymethylfurfural (HMF) to renewable fuels such as 2,5-dimethylfuran (DMF) and 2,5-dimethyltetrahydrofuran (DMTHF) is of significance for sustainable energy supply. For efficient catalyst design, it is important to understand the catalytic behavior and clarify the influence of physico-chemical properties of catalyst on reaction performance. Herein, to study the structure-activity relationships of monometallic Cu catalysts for HMF hydrogenolysis, a series of Cu/SiO All the chemicals were used without further purification.HMF was purchased from Shanghai Mode Pharmaceutical Technology Co., Ltd. Tetrahydrofuran (THF) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. 2,5-dimethylfuran (DMF), tridecane was obtained from Aladdin Reagent (Shanghai) Co., Ltd. SiO2 catalysts were prepared by three different method, namely excessive impregnation (EI), deposition-precipitation (DP) and hydrothermal (HT) method. For a typical EI method, 1.89 g CuNO3•3H2O was dissolved in 70 ml deionized water, and then 1.0 g SiO2 was added into the solution, stirred at room temperature for 12 h. After that, the suspension was dried at 60°C for 12 h. The obtained precursor was then calcined in static air at 450°C for 4 h, and then reduced in flow 10% H2/Ar mixture at 450°C for 6 h before use. The catalyst was denoted as Cu/SiO2-EI.Cu/SiO3•3H2O was dissolved in 70 ml deionized water, and then silica sol  was dropped into the solution and stirred for 0.5 h. Then 1.0 mol/L NaOH solution was dropped into the above mixture with stirring, until the pH of the suspension to a set value of 9. Subsequently, the obtained suspension was filtered, washed by deionized water and dried at 60°C for 12 h. The obtained precursor was then calcined and reduced with the same procedure as EI method. The catalysts were denoted as Cu/SiO2-DP.For a typical DP method, 1.89 g CuNO3•3H2O was dissolved in 70 ml deionized water, and then a certain amount of NH4Cl, NH3·H2O and silica sol  were added into the solution to reach a set pH value (6.5 or 8.5) and stirred for 0.5 h. After that, the mixture was placed in ultrasonic bath for 0.5 h, and then transferred into a 100 ml Teflon-lined autoclave and aged at 120°C for 4 h. After cooling to room temperature, the suspension was filtered, washed by deionized water and dried at 60°C for 12 h. The obtained precursor was then calcined and reduced with the same procedure as EI method. The catalysts were denoted as Cu/SiO2-HT-6.5 or Cu/SiO2-HT-8.5.For a typical HT method, 1.89 g CuNOλ = 0.1541 nm) operating at 40 kv and 40 mA. The crystal size was calculated using the Scherrer equation through the Cu 111) diffraction peak.The X-ray diffraction (XRD) patterns of the catalysts were recorded by a Bruker D8 Foucus X-ray diffractometer with Cu Kα radiation  method.N2 temperature programmed reduction (H2-TPR) was performed on a Huasi DAS-7200 automatic chemisorption apparatus. The samples were pretreated with pure Ar at 150°C for 1 h before reduction. After cooling to room temperature, 10% H2/Ar mixture was introduced and the sample tube was heated to 800°C at a heating rate of 10°C/min. TCD was used as the detector to collect signals.HTransmission electron microscopy (TEM) images were taken using a JEOL 2100F microscope operated at an acceleration voltage of 200 kV. The sample was grinded and dispersed in ethanol under supersonic waves, and then dropped on a copper net and dried before use.The copper content of the catalyst was determined by Agilent 725ES inductively coupled plasma atomic emission spectrometer (ICP-AES).X-ray photoelectron spectroscopy (XPS) was tested on a Thermo Scientific Escalab 250Xi X-ray photoelectron spectrometer with a monochromatic Al Kα radiation . All of the binding energies were calibrated by C 1s and the binding energy of C-C is 284.6 eV.2 for three times, the reactor was conducted under H2 pressure at the required condition for a desired time with magnetic stirring. After the reaction, the reactor was cooled with ice-water and depressurized carefully. After centrifugation of the catalyst, the liquid products were identified by GC-MS  equipped with an HP-5 capillary column and quantitatively analyzed by GC (Agilent GC7890 B) equipped with a flame ionization detector (FID) and an HP-5 capillary column using tridecane as an internal standard. The injector temperature was set at 270°C, and the column temperature was increased from 50 to 200°C with a ramp rate of 10°C/min.The hydrogenolysis of HMF was carried out in a batch reactor equipped with a magnetic stirrer. Typically, 0.05 g HMF, 0.05 g catalyst, 100 μL tridecane  and 5 ml tetrahydrofuran (THF) were put into a Teflon-lined stainless-steel autoclave (50 ml). After purging with H2 catalysts were prepared by impregnation (EI), deposition-precipitation (DP) and hydrothermal (HT) methods at different pH value. 2-EI and Cu/SiO2-DP catalysts exhibit diffraction peaks at 35.5°, 38.7°, 48.7°, 53.5°, 61.5° and 66.2°, indexed to (110), (11–1), (111), (20–2), (020) and (11–3) planes of CuO, suggesting the presence of bulk CuO. Interestingly, obvious CuO diffraction peaks were observed for Cu/SiO2-HT-6.5, but none of them was found for Cu/SiO2-HT-8.5, which indicates CuO species were highly dispersed on the latter catalyst. The high dispersion of CuO species on Cu/SiO2-HT-8.5 can be resulted from the calcination of layered copper silicate , (200) and (220) planes of Cu  diffraction peak, which suggests an agglomeration of Cu cluster. The Cu characteristic diffraction peaks can also be clearly found on Cu/SiO2-DP and Cu/SiO2-HT-6.5 catalysts, while no obvious Cu diffraction peaks observed on Cu/SiO2-HT-8.5, indicating the high dispersion of Cu nanoparticles on the latter catalyst. Cu/SiO2-HT-6.5 still exhibits the diffraction peaks at 35.5° (110) and 38.8° (11–1) of CuO, suggesting an incomplete reduction of the catalyst. In addition, Cu/SiO2-DP and Cu/SiO2-HT catalysts all exhibit the diffraction peaks at 36.5°, 42.3° and 61.3°, attributed to the (111), (200) and (220) planes of Cu2O  and pore volume (0.92 cm3g−1), while Cu/SiO2-EI shows the smallest ones . The large BET specific surface area of Cu/SiO2-HT-8.5 allows the high dispersion of CuOx species on the SiO2 surface, consistent with the XRD results. The Cu contents in Cu/SiO2-EI and Cu/SiO2-HT catalysts analyzed by ICP-AES were close to the set value (33 wt%), whereas only 23 wt% Cu was detected in Cu/SiO2-DP catalyst, which indicates almost a third of Cu content was lost during the DP process.N2 catalysts were investigated by H2-TPR experiments. As shown in 2-TPR profile of Cu/SiO2-EI and Cu/SiO2-DP presents a broad hydrogen consumption peak centered at 275 and 284°C, respectively, which can be attributed to the reduction of bulk CuO  and high binding energy (933.7∼934.1), indicating that Cu element in these three catalysts exists not only as Cu+ or Cu0, but also as Cu2+ species  (0/(Cu0+ Cu+) varies from 0.40 to 0.59 as the different synthetic method of Cu/SiO2, with the Cu/SiO2-HT-8.5 catalyst showing the largest proportion of Cu0. It is known that the weaker interaction between Cu species and the support leads to the easier reduction of CuO to Cu0, while the stronger interaction generates more Cu+ species. The largest proportion of Cu0 in Cu/SiO2-HT-8.5 catalyst indicates that the weakest metal-support interaction compared with the other three catalysts, which is consistent with the H2-TPR results.Since it is difficult to distinguish the Cu913.0 eV , correspectively . Accordiu0+ Cu+) . The pea2 catalysts prepared by different methods in HMF hydrogenolysis is shown in 2-EI) and deposition precipitation method (Cu/SiO2-DP) gave 2,5-dihydroxymethylfuran (BHMF) as a main product with 83.1 and 84.9% yield, respectively, while the total yields of 2.5-dimethylfuran (DMF) and further hydrogenation product 2.5-dimethyltetrahydrofuran (DMTHF) were less than 10%, suggesting a poor hydrogenolysis activity. In contrast, Cu/SiO2 prepared by hydrothermal method offered a much stronger hydrogenolysis activity with DMF and DMTHF as the majority products, especially for Cu/SiO2-HT-8.5 which achieving the highest activity in HMF hydrohydrolysis with 57.1% yield of DMF and 34.5% yield of DMTHF. It was reported that the activity of Cu-based catalysts in HMF hydrogenolysis is proportional to the specific surface area of the metallic Cu0. The high activity for HMF hydrogenolysis over Cu/SiO2-HT-8.5 catalyst can be attributed to the high Cu dispersion, small Cu particle size and high proportion of Cu0, which is well evidenced by XRD characterization, H2-TPR, TEM images, XPS and XAES spectra.The reaction activity of Cu/SiO2-HT-8.5 was studied by analyzing the product distribution at different reaction time, as shown in 2-HT-8.5 catalyst, confirming the high hydrogenation activity of Cu species. It is noted that no 5-methylfurfural (5-MF) was detected, revealing that HMF was first undergo hydrogenation of aldehyde group to BHMF rather than hydrogenolysis of hydroxyl group to 5-MF. After reaction for 4 h, the yield of DMF gradually increased to 29.8%, with the decrease of BHMF to 41.8% and slight increase of HMMF to 13.0%. Only trace of DMTHF was produced at this time, indicating that the hydrogenolysis of hydroxyl group is much faster than the hydrogenation of furan ring over Cu/SiO2-HT-8.5. As the reaction further extended to 8 h, the yield of DMF continued to increase to 58.6%, with the complete conversion of BHMF and HMMF. At the same time, it is noteworthy that the yield of DMTHF also increased rapidly to 30.2%, as well as a small amount of hydration ring opening products of DMF. When the reaction was further extended to 12 h, the yield of DMF decreased from 58.6 to 49.8%, and the yield of DMTHF further increased to 38.8%, indicating that the further extension of reaction time would promote the cyclic hydrogenation reaction of DMF to DMTHF. During the reaction period, small amount of other by-products  were also detected, totally account for ca. 10%。According to the variation tendency of product distribution versus reaction time, the reaction pathways of HMF hydrogenolysis to DMF and DMTHF over Cu/SiO2-HT-8.5 was proposed in The reaction pathway of HMF hydrogenolysis over Cu/SiO2 for HMF hydrogenolysis, we investigated the influence of reaction temperature and H2 pressure on the reaction performance, as shown in oC at 0.6 MPa H2 pressure, the yield of DMF first increased from 47.5 to 57.1% and then decreased to 51.0%. According to the detection of the product distribution, lower temperature (180oC) led to the incomplete conversion of intermediate HMMF and higher temperature (220oC) resulted in a small amount of side reaction of DMF to hydration by-products. At the same time, the yield of DMTHF continuously increased from 30.7 to 36% with the increase of reaction temperature, indicating that higher temperature accelerated the reaction rate of furan ring hydrogenation and in favor of the production of DMTHF.To further tailor the catalytic performance of Cu/SiOoC, the variation of H2 pressure showed that the H2 pressure for HMF hydrogenolysis can be as low as 0.2 MPa, at which a high DMF yield of 66.8% was reached, along with 24.3% yield of DMTHF. This result further confirms the high activity of Cu/SiO2-HT-8.5 catalyst for HMF hydrogenolysis. Increasing of the H2 pressure from 0.2 to 1.4 MPa led to the decrease of the DMF yield from 66.8 to 46.5% with a slight increase of DMTHF, indicating that high H2 pressure in favor of the hydrogenation of furan ring, leading to a decline of DMF yield.When the reaction temperature was fixed at 2002-HT-8.5 catalyst for HMF hydrohydrolysis was also investigated under the optimal reaction conditions . After the first run, the catalyst was separated from the liquid phase by centrifugation, washed with THF, dried and re-used directly for the second run. No obvious activity loss was found after five successive runs and the total yield of DMF and DMTHF maintain close to the values as the first run (2-HT-8.5 catalyst. These results suggest that the high dispersion of Cu species with smaller Cu particle offers not only high activity for HMF hydrogenolysis, but also high catalyst stability.The stability of a catalyst is an important factor to evaluate its prospect for industrial application. Therefore, the cycle stability of Cu/SiOirst run , reveali2 catalysts prepared by different method for HMF hydrogenolysis. It was demonstrated that Cu/SiO2-HT-8.5 catalyst prepared by hydrothermal method showed the best catalytic activity in HMF hydrohydrolysis reaction. Under the optimal reaction condition, the total yield of liquid fuels reaches 91.6% with 57.1% yield of DMF and 34.5% yield of DMTHF in THF solvent. A combination of multiple characterization revealed that the Cu particles in the Cu/SiO2-HT-8.5 catalyst have uniform size and high dispersion. The Cu species and the SiO2 support have relatively weak interaction and are easy to be reduced to Cu0, which makes it an excellent catalyst for the hydrogenolysis of HMF. This work provides a new possibility for cheap monometallic catalyst design for biomass valorization.In summary, we have studied the structure-activity relationships of four Cu/SiO


A clinical utility index was used to assess the appropriateness of the selected dose.Logistic models and Cox regression analyses were applied to correlate lurbinectedin exposure metrics  logistic model, where Emax was dependent on chemotherapy-free interval (CTFI). Cox regression analysis with OS found relationships with both CTFI and AUCu. An Emax logistic model for G4 neutropenia and a linear logistic model for G ≥ 3 thrombocytopenia, which retained platelets and albumin at baseline and body surface area, best fitted to AUCtot and AUCu. AUCu between approximately 1000 and 1700 ng·h/L provided the best benefit/risk ratio, and the dose of 3.2 mg/m2 provided median AUCu of 1400 ng·h/L, thus maximizing the proportion of patients within that lurbinectedin target exposure range.Effect of lurbinectedin AUC2 q3wk.The relationships evidenced in this integrated E–R analysis support a favorable benefit-risk profile for lurbinectedin 3.2 mg/mClinicaltrials.gov: NCT02454972; registered May 27, 2015.The online version contains supplementary material available at 10.1007/s00280-021-04366-3. Lung cancer is the leading cause of cancer deaths worldwide. Small-cell lung cancer (SCLC) accounts for about 15% of lung cancers overall and is a particularly aggressive neoplasm with a 5-year survival rate of < 5%. Little progress has been made in improving the outcome for patients with this malignancy over the past 30 years. When patients with SCLC relapse, few therapeutic options are available. Until recently, topotecan was the only approved drug for second-line treatment of patients with a chemotherapy-free interval (CTFI) longer than 60 days. However, topotecan use is challenging because of its associated hematological toxicities and relatively modest clinical benefit  ) and sensitive disease (11.9 months [95% CI 9.7–16.2]). In the responders, median duration of response was 5.1 months (95% CI 4.9–6.4 months). Based on these results, on June 15, 2020, the US Food and Drug Administration (FDA) granted accelerated approval to lurbinectedin monotherapy in SCLC that has relapsed from platinum compound-based first-line chemotherapy  vs. sensitive disease [≥ 90 days]) was included as a prognostic factor, and OS was a secondary endpoint.The E–R analysis of safety included G4 neutropenia as a primary endpoint since it is the main dose-limiting toxicity associated with lurbinectedin. G4 neutropenia events generally involve the administration of G-CSF and eventually evolve to febrile neutropenia, a life-threatening adverse event. G ≥ 3 thrombocytopenia was included as a secondary endpoint; although occurrences are less frequent than G4 neutropenia, thrombocytopenia may lead to bleeding events and involve the administration of platelet transfusions.u than AUCtot. For E–R analysis of efficacy (ORR and OS), patients initially were classified into four equal-size groups based on the quartiles of AUCu. For ORR and safety outcomes, several mathematical models relating lurbinectedin AUCu to the probability of response, G4 neutropenia and G ≥ 3 thrombocytopenia, were fitted in NONMEM. Initially, continuous AUCu was assumed to be linearly related to the ORR logit and, if deemed appropriate, additional non-linear functions were used, including maximum lurbinectedin effect (Emax) and sigmoid-Emax logistic regression models. Odds ratios for either event were calculated. The final E–R models of safety (G4 neutropenia and G ≥ 3 thrombocytopenia) were used to run simulations (N = 100 replicates) to predict the effect of dose regimen modifications. For OS, Cox proportional-hazards models relating AUCu to the hazard of death were developed.Multivariate analyses were performed to simultaneously incorporate into the models the relationship between AUC and the significant prognostic factor , as well as describe the predictive factors  identified in previous exploratory and univariate analyses, which showed a better correlation of AUCThe relationship between the probability of ORR and the probability of G4 neutropenia was estimated by means of a CUI , a quant2) with two dose regimens (day 1 q3wk and days 1 and 8 q3wk), is described in Eqs. The final model, using 9176 total plasma concentrations from 755 patients treated at a wide range of lurbinectedin doses (0.02–6.9 mg/mt1/2) of about 8–9 min is followed by a slower t1/2 of about 1.9 h and a terminal t1/2 of about 51 h, with the latter phase constituting a substantial portion of the overall AUC. In study B-005, the median (range) of estimated lurbinectedin AUCtot and AUCu at 3.2 mg/m2 in study B-005 were 493 (176–1083) μg·h/L and 1400 (433–3136) ng·h/L, respectively.An open three-compartment model with linear elimination was able to describe the time course of total plasma concentration and its variability. An initial rapid distribution half-life  in the final model are shown in Table u above 1337 and 1433 ng·h/L.Among several linear and non-linear models examined, a sigmoid-eter Eq. , was foumentclass2pt{minimmentclass2pt{minimmentclass2pt{minimu and differences in OS between sensitive and resistant disease, upper quartiles (second to fourth) were pooled and compared against the lowest quartile and stratified by CTFI  showed statistically significant differences. Median overall survival in patients with resistant disease were 3.81 months (95% CI 0.92–4.37) and 6.24 months (95% CI 4.27–8.08) for first and upper quartiles, respectively, after the start of lurbinectedin treatment, while in patients with sensitive disease were 7.26 months (95% CI 3.22–7.79) and 15.90 months (95% CI 10.87–19.29), respectively  and 0.41 (95% CI 0.23–0.72), respectively (Table u (μg·h/L), in patients with resistant disease than in patients with sensitive disease.Based on the differences between first quartile and upper quartiles of AUCTFI Fig. . In patiEmax model based on changes of AUCu, where the base parameter was fixed to a low value (− 10), was finally selected. An equivalent model with AUCtot presented higher objective function value (ofv), although this improved when AAG was included.Several covariates were explored to assess their impact on G4 neutropenia: dose, neutrophils at baseline, albumin, AAG, and BSA. After a number of models were tested using continuous metrics for exposure, an u. Parameter estimates with RSE in the final models based on AUCu and AUCtot are shown in Table Figure u from study B-005 was bootstrapped to predict the incidence of G4 neutropenia at doses of 3.2, 2.6, and 2.0 mg/m2, which correspond to the recommended dose, the first and second 20% dose reductions, as recommended in the label. The resulting decreases in lurbinectedin exposure following the first and second 20% dose reductions are expected to lower the incidence of G4 neutropenia from the 16.5% (95% CI 16.4–16.6) incidence at 3.2 mg/m2 to 11.3% (95% CI 11.2–11.4), and 7.5% (7.4–7.7), at 2.6, and 2.0 mg/m2.AUCu showed the best fit, where higher values of retained covariates reduced the likelihood of G ≥ 3 thrombocytopenia.Plateletsi, BSAi, and Albumini correspond to the individual values for AUCu, platelets, BSA, and albumin at baseline, respectively, for the ith individual. An equivalent model based on AUCtot showed a higher ofv, which improved when AAG was added  were examined. A linear logistic model based on AUCmentclass2pt{minimCL in the PopPK model, although it did to V1, V2, V3, and Q3, confounding factors causing the effect of BSA on thrombocytopenia were explored. In this sense, factors included in a previously developed semi-mechanistic PKPD model of lurbinectedin on platelets [50), baseline platelets  and tumor type (affecting EC50), with pancreas and ovarian as more sensitive tumors, were the detected covariates. BSA was neither related with baseline platelets nor with tumor type, so confounders affecting the relationship between BSA and thrombocytopenia are unlikely.Since BSA was not associated to latelets  were anau from study B-005 was bootstrapped to predict the incidence of G ≥ 3 thrombocytopenia at doses of 3.2, 2.6, and 2.0 mg/m2. The resulting decreases in lurbinectedin exposure following the first and second 20% dose reductions are expected to lower the incidence of G4 neutropenia from the 4.8% (95% CI 4.7–4.9) incidence at 3.2 mg/m2 to 3.5% (95% CI 3.4–3.5), and 2.75% (2.6–2.7), at 2.6, and 2.0 mg/m2.As described above for neutropenia, AUC2 for a BSA of 1.75 m2) increased a 16% in patients lower BSA. Less variation in the incidence of G ≥ 3 thrombocytopenia was observed with BSA-based dosing: 6.4% (95% CI 5.3–8.1) in patients with lower BSA (< 1.64 m2) and 3.1% (95% CI 1.9–4.8) in patients with larger BSA; with FD, the incidences were 7.6% (95% CI 6.2–9.5) and 2.5% (95% CI 1.6–4.0), respectively.As BSA was retained in the model, the incidence of G ≥ 3 thrombocytopenia at a fixed dose of 5.6 mg  and 19.3%  for resistant and sensitive SCLC patients, respectively. Therefore, based on these point estimates, lurbinectedin AUCu above 1072 ng·h/L and 1003 ng·h/L, which are similar to EC50 of ORR  suggest mean ORRs of 7.5%  treated at a wide range of lurbinectedin doses, although model diagnostics showed a better fit of AUCu over AUCtot. However, the efficacy model could not be fitted to AUCtot, but only to AUCu. Given the low extraction ratio, lurbinectedin AUCtot and unbound fraction were interfered by plasma protein levels, which vary substantially among cancer patients, while AUCu remained unaffected [u is more representative of active lurbinectedin AUC, thus exposing existing relationships with efficacy outcomes, regardless of the smaller efficacy database of patients (n = 99) treated at a single dose level.In this sense, the safety models presented herein established E–R relationships with both AUCaffected . AUCu is2 q3wk. One or two sequential 20% dose reductions during the course of lurbinectedin treatment are adequate to manage patients who develop severe episodes of myelosuppression.The relationships evidenced in this integrated E–R analysis, with efficacy variables at a single dose level and safety variables at a wide range of doses, support a favorable benefit-risk profile for the approved lurbinectedin dose regimen of 3.2 mg/mSupplementary file1 (PDF 202 KB)Supplementary file2 (DOCX 13 KB)Below is the link to the electronic supplementary material.


N-((4-acetylphenyl)carbamothioyl)pivalamide (3) was synthesized by inert refluxing pivaloyl isothiocyanate (2) and 4-aminoacetophenone in dry acetone. The spectroscopic characterization  and single crystal assays determined the structure of synthesized compound (3). Systematic experimental and theoretical studies were conducted to determine the molecular characteristics of the synthesized crystal. The biological examination of (3) was conducted against a variety of enzymes i.e., acetyl cholinesterase (AChE), butyl cholinesterase (BChE), alpha amylase, and urease enzyme were evaluated. The crystal exhibited approximately 85% enzyme inhibition activity against BChE and AChE, but only 73.8 % and 57.9% inhibition activity against urease and alpha amylase was observed respectively. The theoretical calculations were conducted using density functional theory studies (DFTs) with the 6–31G  basis set and B3LYP functional correlation. The Frontier molecular orbital analysis revealed that the HOMO/LUMO energy gap was smaller, which corresponds to the molecule’s reactivity. In terms of reactivity, the chemical softness value was found to be in good agreement with experimental values. In Crystal structure analysis, the intramolecular N—H•••O hydrogen bond generates a S 6) ring motif and N—H•••O interactions exist in crystal structure between the centroids of neighboring parallel aromatic (C4-C9) rings with a centroid to centroid distance of 3.9766 (7)Å. These intermolecular interactions were useful in structural stabilization. The Hirshfeld surfaces and their related two-dimensional fingerprint plots were used for thorough investigation of intermolecular interactions. According to Hirshfeld surface analysis of the crystal structure the most substantial contributions to the crystal packing are from H ••• O and H ••• N/N ••• H interactions. Molecular docking studies were conducted to evaluate the binding orientation of synthesized crystal with multiple targets. The compound exhibited stronger interactions with AChE and BChE with binding energies of -7.5 and -7.6 kcal/mol, respectively. On the basis of in-vitro and in-silico findings, it is deduced that N-((4-acetylphenyl)carbamothioyl)pivalamide 3) possesses reactive and potent multiple target inhibitory properties.The crystal structure of  N-heterocyclic compounds such as dyes, antibiotics, vitamins, and agrochemicals have depicted a wide variety of pharmacological and physiological properties .Given the therapeutic relevance of the thiourea scaffold, it is necessary to examine its molecular structure, non-covalent interactions, thermodynamic and electrical characteristics in order to determine the impact of various substituents on the biological activity of the parent chemical. In this context, DFT investigations are an effective method for establishing a correlation between theoretical and experimental compound properties. These investigations offer adequate information about the compound’s molecular structure, electronic and thermodynamic characteristics. These methods have the potential to determine the molecular characteristics of a substance with precision and accuracy. . The exi process . HoweverWe have previously reported the N-((4-acetylphenyl) carbamothioyl)-2,4-dichlorobenzamide derivative  as a pos1H-NMR spectra were collected in deuterated solutions using a Bruker 300 MHz spectrometer with TMS as the internal standard. A13C NMR spectrum was generated using a 75 MHz instrument. Chemical shift (CS) values are measured in parts per million (ppm). A GC-MS 6890N equipment from Agilent Technologies was utilized to record the mass spectrum . On an aluminum plate precoated with silica gel, thin-layer chromatography (TLC) was performed . UV light was utilized for chromatogram visualization (254 and 260 nm).The melting point was determined without correction using a Gallenkamp melting point device (MP-D). The infrared spectra were recorded using KBR pellets on a Shimadzu IR 460 spectrometer. The The potassium thiocyanate (10 mmol) was dropwise added to the pivaloyl chloride (5.0 mmol) in dry acetone (20 ml) and the mixture was refluxed for three hours. The reaction mixture was then allowed to cool to room temperature. The reaction mixture was then refluxed with a solution of 4-amino acetophenone (5 mmol) in dry acetone for 24 h. TLC was employed to monitor the development of the reaction. Upon completion of the reaction, the reaction mixture was poured over crushed ice and the product thiourea 3) was precipitated, filtered, and recrystallized with ethanol.f: 0.63, Ethyl acetate: n-Hexane (4:6); FTIR (cm−1): 3,233 (NH Stretch), 2,955 (sp2 CH-Ar), 2,936 (sp3 CH), 1,655 (C=O), 1,525 (C=O), 1,174 (C=S); 1H-NMR; : δ 12.95 , 9.40 , 8.06–7.96 ,2.89 ; 1.38 ; 13C-NMR : δ 195.91 (C=O); 180.25 (C=S); 178.8, (C=O); 142.1, 134.6, 128.9 122.8(Ar-C); 40.1 (C), 29.7 (3*CH3), 25.7 (CH3).Light Green solid; M.P: 140°C; Yield: 79% Ra-amylase enzyme solution, 10 μl starch, 10 μl of 3) with 200, 100 and 50ppm concentration was mixed in 96-well plates. All the plates were incubated at 50°C for 30 min. Then 100 µl of HCl (0.2 M) was added as stopping reagent followed by addition of 100 µl of iodine reagent (5 mM KI and 5 mM I2). The urease inhibition activity of 3) was carried out by the previously reported method  was performed using previously reported method . For expd method . For expn assays  were carKα radiation (λ = 1.54184 Å). The same methodology was employed as reported in our previous study  equipped with Cu us study . The deti and De, respectively, while Dnorm was used to denote the normal contact distance. Dnorm has been spelled in white, red, and blue. The CIF-formatted input file is retrievable from the supplemental materials.Hirshfeld surface (HS) analysis is the most effective method for determining crystal structure interactions . Using CThe DFT studies were conducted to optimize the molecular geometry of the synthesized derivative . The strinvitro studies. This was done to link our in-silico results with the in-vitro analysis. The compound 3, was subjected to in-vitro and in-silico testing against four different targeted proteins, i.e., AChE, BChE, α-amylase, and urease. Initially, the crystallographic structures of all proteins were retrieved from the protein data bank (www.rcsb.org) using PDB IDs 2QMK, 4AC7, 7RB5, and 6EP4 respectively. All these proteins were prepared using the AutoDock suite, which included the removal of het and water molecules, the addition of polar hydrogen and kollman charges. Moreover, missing residues were also prepared. After preparation of targeted proteins, the 2D structure of Ligand was generated by using Chemdraw  carbamothioyl) pivalamide is shown in 1H NMR, 13C NMR, and FT-IR spectroscopy.A synthetic procedure for −1 aromatic stretch 2,955 cm−1 (sp2 CH-Ar), aliphatic (sp3 CH) at 2,936 cm−1 and C=O at 2,955 cm−1. The 1H NMR spectrum of derivative 3 showed two characteristic signals of N-H at δ 12.95 and 9.40 ppm. Two multiplet for four aromatic protons appeared at δ 8.06–7.96 ppm, singlet for nine protons of CH3 at δ 1.38, and a singlet for CH appeared at δ 2.89 ppm. In 13C NMR, three characteristic signals, one for thiocarbonyl and two for carbonyl carbons were observed. These signals appeared at δ195.91 for C=O, δ180.25 for C=S and δ178.8, ppm for another C=O). Four carbons of aromatic system observed at δ142.1–122.8ppm. Three signals for aliphatic carbons observed in the range δ 40.1–25.7 ppm.The FT-IR spectrum displayed a characteristic stretching vibration for the NH functionality at range 3,233 cm50 values of 26.23 and 30.9 ppm, respectively. However, IC50 values against alpha amylase and urease enzymes were marginally higher (160.33 and 91.5 ppm respectively)  .%inhibi50 value 91.5 ± 3.9 ppm. Finally, against a-amylase comparatively lower activity was recorded with IC50 value 160.33 ± 1.7 ppm. Following the experimental findings compound 3 was docked with all enzyme proteins to identify the possible mode of interactions responsible for their activities and results are summarized in Secondly, compound 3 has moderate activity against urease with ICSimilar to enzyme inhibition experiments, docking results showed remarkable energies in order of BChE > AChE > Urease > alpha-amylase. Both acetylcholinesterase and butyrylcholinesterase receptors exhibited robust interactions with the ligand compound 3 at energies of −7.5 kcal/mol and −7.6 kcal/mol, respectively. Similarly, alpha-amylase and urease exhibited comparable and significant interactions with compound 3, with binding energies of −6.1 and −6.3 kcal/mol, respectively. The interactions present in docked ligand receptors include Vander Val forces, ionic and hydrogen interactions. The bond energy are low and bond lengths are less than 4 Angstroms which indicate a good interaction. These interactions take place between charged groups on ligand and different amino acid residues of the protein that are described in the in-vitro link can be obtained.Our docking studies corroborated the experimental findings showing the potential mechanism of action (bond length) of chemical 3 with distinct receptors in close proximity . Figure The intramolecular N—H•••O hydrogen bond generates a S 6) ring motif in the title molecule . N—H•••OCrystal Explorer 17.5 was used to conduct a Hirshfeld surface (HS) investigation to visualize the intermolecular interactions in the crystal of the title chemical (3). The outcomes of the HS analysis are shown in the figures below ,7,8,9,10ele), polarisation (Epol), dispersion (Edis), and exchange-repulsion (Erep) energies with scale factors of 1.057, 0.740, 0.871, and 0.618, respectively, is the total intermolecular energy (Etot).The intermolecular interaction energies are calculated using the CE–B3LYP/6–31G  energy model in Crystal Explorer 17.5, which generates a cluster of molecules by applying crystallographic symmetry operations with respect to a central molecule within a radius of 3.8 by default. The sum of electrostatic (Eele (red cylinders), Edis (green cylinders), and Etot (blue cylinders) each have their own energy system  carbamothioyl) pivalamide was synthesized and characterized by using spectroscopic and single crystal assays. DFT analysis revealed the reactive nature of the compound with a narrow LUMO/HOMO energy gap. The narrow energy gap between Frontier orbitals is predicting the reactive nature of (3). Hirshfeld analysis provided significant insight into intermolecular and non-covalent interactions existed among crystal structure. According to Hirshfeld surface analysis of the crystal structure the most substantial contributions to the crystal packing are from H ••• O and H ••• N/N ••• H interactions. X-ray refinement data demonstrated that C—H•••O and N—H•••O contacts stabilized molecule geometry, whereas hydrophobic interactions including van der Waal forces also stabilized the crystal packing. The in-vitro enzyme inhibition assay was conducted to evaluate the multi-targeting nature of title molecule. The crystal exhibited approximately 85% enzyme inhibition activity against BChE and AChE and 73.8 % and 57.9% inhibition activity against urease and alpha amylase respectively. Moreover, in-silico binding orientation of crystal was assessed through molecular docking approach. It was observed that compound 3 produced stable molecular interactions against all targeted enzymes particularly AChE and BChE with docking score of −7.5 and −7.6 kcal/mol respectively. Moreover, the electrostatic surface potential map reveals that atoms N1 and N2 are electron donors, whereas atoms O1 and O2 are electron acceptors. On the basis of the results, it was determined that compound 3 featured a reactive profile and powerful anti-enzyme activity, making it a potential new therapeutic strategy against AChE and BChE-associated malignancies.In the current study,


The hexagonal metallocycle units are assembled into a densely packed ABCABC… sequence (like the fcc geometry) to construct one-dimensional (1D) helical π-stacked columns and 1D pore channels, which were maintained under the liberation of H2O molecules. The NDI cores were partially reduced to form radicals as charge carriers, resulting in a room-temperature conductivity of (1.2–2.1) × 10−4 S cm−1 (pressed pellet), which is superior to that of most NDI-based conductors including metal–organic frameworks and organic crystals. These findings open up the use of metallocycles as building blocks for fabricating conductive porous molecular materials.Electrical conduction among metallocycles has been unexplored because of the difficulty in creating electronic transport pathways. In this work, we present an electrocrystallization strategy for synthesizing an intrinsically electron-conductive metallocycle, [Ni −4 S cm−1 in polycrystalline pellet.Intrinsically electron-conductive metallocycle was synthesized. π-Radicals play a key role in constructing π-stacked columns among molecular hexagons and achieving high electrical conductivity over 10 For instance, anticancer ability,15 drug delivery,16 chirality recognition,17 and other molecular responsive functions3,11,18 have been realized by the flexible cavity of metallocycles. Furthermore, organic linkers with unique electronic properties have been key to fabricating functional metallocycles such as luminescent liquid crystals,19 asymmetric catalysts,20 and artificial light-harvesting systems.21 Although some metallocycles are not isolable in solution, solid-state functions have also been a subject of active research recently. To date, single-molecule magnet behavior,10,22 solid-state luminescence,23 heterogeneous catalytic behavior,24 gas storage,25etc., have been reported in crystalline metallocycle assemblies. In this context, engendering electron transfer between assembled metallocycles is promising for providing conductivity as a new function of metallocycles, and moreover, it can be an alternative approach to design conductive porous crystals such as metal–organic frameworks (MOFs).26 However, only the hole mobility of dipyrrin-based polygons has been reported as the charge-transport property in metallocycles.27 Intrinsic conductivity, which requires both rich charge carriers and through-space conduction pathways among metallocycles, has not been realized thus far.Giant macrocycles have attracted a great deal of attention due to their distinctive molecular structures and diverse functions.28 and, more recently, porous molecular conductors (PMCs),29,30 which are porous crystals consisting of linear coordination polymers with a conductive π-stacked array of N,N′-di(4-pyridyl)-1,4,5,8-naphthalenetetra-carboxdiimide (NDI-py) linkers. The NDI core is a well-known electron acceptor used in various research fields31 such as self-assembly,32 molecule sensors33 and molecular electronics,34 whereas its application in intrinsic electron conductors is still in the early stage of development.Electrocrystallization is a reasonable method to fulfill the above requirements, because the unpaired electrons  generated on π-conjugated planes by a redox reaction can be charge carriers and also induce an attractive interaction between π-conjugated planes. It has been widely used for the syntheses of organic conductors35 and metallocycles.36Herein, we report an electrically conductive metallocycle synthesized by electrocrystallization for the first time. The coordination-driven self-assembly of hexagonal metallocycle units and the columnar self-assembly of π-conjugated organic linkers occurred together in the electrocrystallization process. In this work, pyrazole was selected as the terminal group of the organic linker because of their preferential construction of various bent coordination polymersN,N′-Di(1H-pyrazol-4-yl)-1,4,5,8-naphthalenetetracarboxdi-imide (NDI-Hpz) and Ni(NO3)2·6H2O were dissolved in N,N-dimethylacetamide (DMA) with a small portion of H2O. Electrocrystallization was carried out by applying a constant direct current of 20 μA to the mixed solution at room temperature (RT). Needle-like tiny black crystals of [Ni6(NDI-Hpz)6(dma)12(NO3)6]·5DMA·nH2O (hereafter abbreviated as PMC-hexagon) were obtained from the cathode in 12 hours.R3̄ (Table S16(NDI-Hpz)6(dma)12(NO3)6] constructed from head-to-tail connection between Ni2+ ions and NDI-Hpz linkers was elucidated as shown in 2+ ion is coordinated by two pyrazole groups, two DMA molecules in cis geometry, and a bidentate NO3− ion. As a result, the Ni2+ ion forms nearly octahedral coordination geometry , resulting in a pucker of the 6-membered metallocycle  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C)⋯H–N hydrogen bonds  analysis revealed that PMC-hexagon crystallized in the trigonal space group  Table S1 with mergeometry . The posllocycle . The slien bonds . Since ic axis. It is constructed from the dense packing of hexagonal metallocycles with an alternating sequence of ABCABC…  .29,30 The transfer integral between the adjacent LUMO of NDI-Hpz linkers along the stacking axis was calculated to be 315 meV. It is significantly larger than those of neutral NDI-based organic semiconductors ,39 reflecting the strong π-stacking interaction in PMC-hexagon.The interplanar distance between adjacent NDI-Hpz ligands along the stacking axis is 3.173 Å (1/3 of the unit cell length c), which is shorter than those (>3.3 Å) between typical neutral NDI cores,2O molecules , whereas their exact number was uncertain because the liberation of H2O molecules occurred even at RT (OH2)4](NO3)1.3±0.1·nDMA (PMC-1).29 However, it is notable that PMC-hexagon retained the crystal structure under the liberation of H2O molecules despite the lack of a coordination polymer network like MOFs. This robustness should originate from the dense packing supported by the strong π-stacking interaction.The dense packing decreases their cavities, resulting in a small void space . On the basis of elemental analysis, the 1D pore channel is likely to accommodate H. S9, ESI. Thus, i3 par channel in the unit cell), PMC-hexagon was more air-sensitive than PMC-1, probably because the fast liberation of H2O molecules from the channel promotes the access of oxygen molecules to NDI˙− species. The nitrogen gas adsorption in the micropore of PMC-hexagon has not been observed  in the electron spin resonance (ESR) spectrum  and the antiferromagnetic interaction and/or partial dimerization along the π-stacked column decreases the total magnetic moment of NDI˙− species. We estimated the number of effective NDI˙− species in the range from 10 to 15% by fitting the temperature dependence of the χMT value (χM: molar spin susceptibility and T: temperature) with the aid of quantum calculations  cores  between two NDI˙− cores and that from NDI˙− to NDI0 cores, respectively.29,30,40,43 The existence of NDI0 species in PMC-hexagon is inconsistent with the formula, which indicates that the charge of NDI-Hpz is −1. Whilst the concentration of NDI0 in the crystal could not be determined with accessible analytical methods, we believe that partial deprotonation of the Hpz group satisfies charge neutrality in this system. The partial absence of the hydrogen-bonded DMA molecules (84% occupancy) may reflect the loss of hydrogen donors due to the partial deprotonation.To investigate the electronic state in detail, the solid-state absorption spectra of PMC-hexagon and NDI-Hpz dispersed in KBr pellets were acquired. The whole process from the synthesis to the measurement was carried out under an argon atmosphere in order to avoid the air oxidation of radicals. Both spectra display strong absorption bands around 3.3 eV, which are assignable to intramolecular π–π* transition in neutral NDI (NDI0) cores .29,30,40σ), the measurements were carried out on 3 mmϕ pressed pellets. The current–voltage (I–V) plots indicate the ohmic characteristic  as shown in σ of PMC-hexagon at RT was calculated to be (1.2–2.1) × 10−4 S cm−1. It is 10 times higher than the σ of PMC-1, which was newly prepared and measured under the same inert conditions in this work  × 10−6 S cm−1)29 was obtained under incompletely inert conditions and is lower than that obtained in the present work ((1.4–2.0) × 10−5 S cm−1 in pressed pellets), indicating that the avoidance of air oxidation is important to properly measure σ in NDI-based conductors. The π-stacked columns in PMC-hexagon are surrounded by Ni complexes and are away from 1D pore channels, freeing it from the perturbation of conduction carriers by disordered molecules in the pores. It may be one of the reasons for the 10-fold increase in conductivity compared to PMC-1. The temperature dependence of σ  is determined to be 0.148 eV by fitting the data with the Arrhenius equation σ = σ0 exp(−Ea/kT), where σ0 is the prefactor, k is the Boltzmann constant and T is the temperature. The Ea of PMC-hexagon is smaller than that in PMC-1 (0.25 eV),29 supporting the highly conducting nature of PMC-hexagon. To date, the reported σ of NDI-based conductive MOFs/coordination polymers29,44 and organic crystals45 have been in the range from 10−7 to 2.0 × 10−5 S cm−1 for pressed pellets and up to 3.3 × 10−3 S cm−1 for single crystals. The σ of a pressed pellet of 1D conductors is typically smaller than that of the single crystal by two to three orders of magnitude. Therefore, PMC-hexagon is one of the most conductive NDI-based crystals. Although the σ of PMC-hexagon is lower than the record values for conductive MOFs with through-space conduction pathways (10−4 to 0.05 S cm−1 for lanthanide-hexahydroxytriphenylene-based MOFs),46 it is higher than that of other π-stacked MOFs (e.g. 10−9 to 7.6 × 10−5 S cm−1 (pressed pellet) for MOFs with a TTF moiety47). Moreover, the high σ (above 0.1 S cm−1) observed in some NDI-based polymers48 implies that we have much room to improve the conductivity of NDI-based metallocycles, which can be an alternative building block to construct conductive porous molecular crystals.The low energy CT band suggests that PMC-hexagon is a narrow-gap semiconductor. Since the single crystals of PMC-hexagon were too small to measure the electrical conductivity (nce of σ  shows th−) provides quite a short interplanar distance (3.173 Å) and conducting carriers, resulting in a high electrical conductivity ((1.2–2.1) × 10−4 S cm−1 at RT) even in the polycrystalline pellet sample. Despite the lack of a coordination polymer network, the dense packing of hexagonal metallocycles provides a robust structure, which was maintained under the liberation of H2O molecules in the pore channels. The elimination of DMA molecules induced the deterioration and disturbed the nitrogen adsorption to pore channels. Although the improvement of the robustness and the conductivity remains to be addressed, the concept of using a metallocycle as a building block provides a new platform for developing electrically conductive porous molecular materials.Intrinsically electron-conductive metallocycle PMC-hexagon was synthesized by using electrocrystallization. The hexagonal metallocycles were densely π-stacked in a face-centered cubic (fcc)-like geometry (ABCABC… sequence) to afford 1D helical π-stacked conductive columns and 1D pore channels. The radical at the NDI core  multisource, YAZAWA CS-12Z, and 0.3 mm electrodes of platinum-iridium alloy wires (Pt : Ir = 80 : 20). All elemental analyses were performed at a J-SCIENCE LAB, JM-11 at the Research and Analytical Centre, Tohoku University. Japan.All purchased chemicals were used without further purification. 2SO4 (98%) (6 mL) and cooled to 0 °C. HNO3 (ca. 67%) (1.2 mL) was then added dropwise over 5 minutes. The mixture was then heated to 60 °C and stirred for 2 hours. The mixture was then cooled to 0 °C, the acidic solution was poured into ca. 100 g of ice and then stirred for 10 minutes upon which, a white precipitate was formed. The mixture was neutralized with Na2CO3(aq), extracted with ethyl acetate (100 mL × 3), washed with brine solution, and concentrated to yield a white crystalline powder. The product was recrystallized from ethyl acetate and dried in vacuo. Yield: 2.059 g (61.2%). 1H NMR : δ 13.94 , 8.87 , 8.25  ppm. Elemental analysis, found: C, 31.95; H, 2.7; N, 37.2. Calc. for C3H3N3O2: C, 31.85; H, 2.7; N, 37.2.Pyrazole  was dissolved in H2 for 1 hour. Pd/C powder (10% w/w) was then added and the mixture was further degassed for 1 hour. The flask was then put under reduced pressure and a H2 gas-filled balloon (1 atm) was then connected. The reaction mixture was then stirred vigorously for 24 hours. The H2 gas was refilled upon depletion during the reaction. Upon completion of the reaction, the Pd/C powder was separated via filtration, and the volume of the filtrate was reduced to yield a reddish oil which partially solidified upon cooling to 5 °C overnight. The product is hygroscopic in ambient air. Yield: 2.04 g (95.5%). 1H NMR : δ 7.28  ppm. Elemental analysis, found: C, 42.1; H, 6.1; N, 48.7. Calc. for C3H5N3: C, 43.35; H, 6.1; N, 50.6. (Note: discrepancy is due to the hygroscopic nature of the product).4-Nitropyrazole  was dissolved in anhydrous EtOH (100 mL) in a Schlenk flask and degassed under N2 and refluxed for 24 hours. Upon cooling to RT, 80 mL of H2O was added to the mixture while stirring to yield a yellow/maroon precipitate. The precipitate was filtered, washed with acetone (30 mL × 3), and dried under air. The product was recrystallized from N,N-dimethylacetamide (DMA) and H2O to yield a light-yellow powder. Single crystals of NDI-Hpz were obtained by slow diffusion of MeOH to a saturated NDI-Hpz solution in DMA. Yield: 1.35 g (43.6%). 1H NMR , 8.71 , 7.95 , 7.63  ppm. Elemental analysis, found: C, 60.15; H, 2.7; N, 21.0. Calc. for C20H10N6O4: C, 60.3; H, 2.5; N, 21.1.4-Aminopyrazole  and 1,4,5,8-naphthalenetetracarboxylic dianhydride  were suspended in DMF (40 mL) and degassed for 20 minutes. The reaction was put under N2 for about 30 minutes. To the solution, Ni(NO3)2·6H2O  and H2O (20 μL) were added and sonicated for around 3 minutes. The solution was then put under a constant current of 20 μA. A black microcrystalline product began to appear around the cathode overnight. After the reaction was continued for about 3 days, the product was carefully removed from the cathode and washed with degassed DMA solution under an N2 atmosphere and dried in glovebox filled with an Ar atmosphere. To obtain single crystals suitable for structural analysis, a degassed DMA solution (3 mL) containing NDI-Hpz , Ni(NO3)2·6H2O  and H2O (20 μL) was prepared. After a constant current of 20 μA was applied to the solution overnight, black needle-like crystals of PMC-hexagon were obtained. Elemental analysis, found: C, 47.2; H, 4.9; N, 16.8. Calcd for C188H237N59Ni6O71: C, 46.9; H, 5.0; N, 17.2 (assumed that n = 12). .For the synthesis of bulk PMC-hexagon, NDI-Hpz  was suspended in DMA (6 mL) and heated until all solids were dissolved. Upon cooling to RT, the solution was degassed under NDetailed data including general information, characterization, crystallographic parameters, magnetism, electrical conductivity and computational methods are available in the ESI.†H. I. conceived and designed the project. M. C. carried out the synthesis, characterization, crystal structure analysis and measuring of the physical properties of PMC-hexagon. R. M. contributed to the design of the experiment, syntheses of ligands and PMC-hexagon, and preliminary crystal structure analysis. Y. S. contributed to the measurement and analysis of electrical conductivity. T. S. and K. U contributed to the magnetic measurements and the analyses using calculation and fitting. K. U. also contributed to the nitrogen adsorption measurement. S. K. performed the theoretical calculation of the transfer integral. T. T. contributed to measurements and characterization related to nitrogen sorption isotherms and the thermogravimetry analysis. S. T., M. Y. and H. I. supervised this work and carried out discussions. M. C. and H. I. wrote the paper.There are no conflicts to declare.SC-013-D2SC00447J-s001SC-013-D2SC00447J-s002


After muscle injury, the interaction between muscle satellite cells (SC) and the immune response is instrumental for the repair and regeneration of skeletal muscle tissue. Studies have reported sex-based differences in the skeletal muscle inflammatory and regenerative response following injury. However, many of these studies investigated such differences by manipulating the concentration of estradiol, in rodents and humans, without directly comparing males to females. We sought to explore differences in the myogenic and inflammatory response following unaccustomed eccentric exercise in males and females. We hypothesized that females would have a blunted myogenic and inflammatory response as compared to males.Methods: 26  healthy young adults (22 ± 0.4 years [mean ± SEM]) performed 300 maximal eccentric contractions (180°/s) of the knee extensors. Muscle biopsies were taken before (pre) and 48 h (post) following eccentric damage. SC content and activation were determined by immunohistochemical and real time-polymerase chain reaction (rt-PCR) analysis. Inflammatory markers were analyzed using rt-PCR.Results: Following eccentric damage, males had a greater expansion of type I-associated SC (p < 0.05), and there was a trend for a greater expansion in total SC (type I + II fibers) (p = 0.06) compared to females. There was a trend for a greater increase in Pax7 and CCL2 gene expression in males compared to females (p = 0.09).Conclusion: We conclude that there are sex-based differences in the myogenic and inflammatory response, where females have a blunted SC and inflammatory response. Understanding the underpinnings of skeletal muscle repair and regeneration are important in developing therapeutic strategies to maximize muscle mass and optimize overall muscle health. The ability to regenerate muscle relies upon a population of resident muscle stem cells, referred to as satellite cells (SC) . Under rWhen skeletal muscle is damaged, inflammatory cells invade the site of injury within 1–6 h . NeutropSeveral human studies , but nothttps://clinicaltrials.gov/as NCT03525197. All participants were recreationally active with no structured exercise in the previous year. Exclusion criteria included smoking, diabetes, the use of nonsteroidal anti-inflammatory drugs (NSAIDs), and/or statins, and history of respiratory disease and/or any major orthopaedic disability. Eleven of the female subjects recruited were normally menstruating, and two were using second-generation oral contraceptives. The study conformed to the guidelines outlined in the Declaration of Helsinki. The participants gave their informed written consent before their inclusion in the study.Twenty-six healthy young males and females (13M/13F) , body composition [dual-energy X-ray absorptiometry (DXA) scan], and a muscle biopsy were taken. Participants were then given a brief period of rest before performing 300 maximal eccentric contractions on the biopsied leg, a protocol often used by our laboratory, known to induce damage in the muscle . 48 h foUnaccustomed eccentric exercise is a common technique that reliably induces a significant level of skeletal muscle damage, evidenced by extensive z-band streaming, desmin disruption , a signiWhole-body lean soft tissue mass (fat-free and bone-free mass), leg lean mass, fat mass, body fat percentage, and bone mineral content were measured  and analyzed  using a DXA scanner, after a 10- to 12 h overnight fast.Two muscle biopsies were taken from the mid-portion of the vastus lateralis under local anaesthetic (1% lidocaine) using a 5 mm Bergstrom needle adapted for manual suction. Both biopsies were performed on a single leg, which was randomized for each participant. One muscle biopsy was obtained at rest (pre) and the other 48 h following eccentric damage (post). Approximately 150 mg of muscle tissue was collected from each biopsy. Following collection of the muscle sample, the muscle was dissected free of adipose and connective tissue and flash-frozen in liquid nitrogen, then stored at −80 °C for later analysis. For immunohistochemistry, a fresh piece of muscle (approximately 40 mg) was sectioned from the biopsies, orientated in cross-section, mounted in OCT compound , and frozen in isopentane cooled with liquid nitrogen. The embedded samples were stored at −80°C and then sectioned (7 μm) at −20°C using a cryostat microtome. The cross-sections were mounted on slides and stored at −80°C for immunohistochemical analysis.Muscle cross-sections (7 µm) were prepared from tissue embedded in optimal cutting temperature compound, air-dried for 30 min, and stored at −80°C. Two immunohistochemical experiments were completed, to analyze SC content for type I and II fibers, and to investigate SC activation. Slides were stained with antibodies against Pax7 , MyoD , A4.951 , and MHC-II . Secondary antibodies used were Pax7 , MyoD , A4.951 , MHC-II , and laminin . Nuclei were labelled with 4,6-diamidino-2-phenylindole  before applying a coverslip with fluorescent mounting media (Dako). Slides were viewed with a Nikon Eclipse Ti Microscope (Nikon Instruments), equipped with a high-resolution Photometrics CoolSNAP HQ2 fluorescent camera (Nikon Instruments). Images were captured and analyzed by using Nikon NIS Elements AR 3.2 software (Nikon Instruments). All images were captured with the 20x objective, and ∼249 fibers per participant per time point were included in the analyses for SC content and activation status. The activation status of satellite cells was determined via the colocalization of Pax7, MyoD, and DAPI . Slides were blinded for both group and time point. All immunofluorescent analyses were completed in a blinded fashion by a single examiner.Muscle samples were homogenized with 1 ml of TRIzol Reagent , in Lysing Maxtrix D tubes , with the FastPrep-24 Tissue and Cell Homogenizer  for a duration of 40 s, at 6 m/s. Following centrifugation, samples were incubated for 5 min to permit complete disassociation of the nucleoprotein complex. Then, 200 µl of chloroform  was added, mixed for 10 s, incubated at room temperature for 3 min, and centrifuged for 15 min at 12,000 g at 4°C. The aqueous phase was transferred into a new tube. RNA was precipitated by adding 500 µl of isopropanol , incubated at room temperature for 10 min, and centrifuged for 10 min at 12,000 g at 4°C. The resultant RNA pellet was resuspended and washed in 1 ml of 75% ethanol , centrifuged for 5 min at 7,500 g at 4°C, and the supernatant was discarded. Finally, the RNA pellet was resuspended and diluted in 50 µl of RNase-free water. RNA concentration and purity (260/280) were determined with a Nano-Drop 1,000 Spectrophotometer . The average RNA purity (260/280) was 1.8 ± 0.1(SEM).Samples were reverse transcribed using a high-capacity cDNA reverse transcription kit , 1000 ng of RNA was diluted in 20 μl reaction solution, as per manufacturer’s instructions. SimpliAmp Thermal Cycler (Thermo Fisher Scientific) was used to obtain cDNA for gene expression analysis.−ΔΔCt method and expressed as fold change from Pre. Due to technical difficulties, only an N = 8 was included for males for CXCL8 gene expression analysis.Quantitative PCR using TaqMan methodology was performed to analyze myogenic , inflammatory , and pan-macrophage cell surface marker (ITGAM/CD11b [Hs00167304_m1]) gene expression. GAPDH (Hs02786624_g1) was used as the reference gene. For each assay, qPCR was performed using 50 ng of cDNA in TaqMan Fast-Advanced master mix on a QuantStudio 5 (Thermo Fisher Scientific) Real-Time PCR system; samples were run in duplicate. mRNA expression was calculated by using the 2t-tests were used to compare differences in baseline characteristics between groups. SC content and activation data were analyzed using a two-way repeated-measures ANOVA with biological sex  as the between-subject factor and time (pre and post) as within-subject factors. Separate ANOVAs were performed for type I, type II, and total fibers. Where significance was found, a Tukey’s post hoc test was performed. Expression of mRNA was analyzed using paired (within-group [time]) or unpaired t-test’s (between-group). Analyses were conducted using SPSS statistical package version 23 .A sample size estimation was conducted  using the following parameters: average effect size of 0.546 on SC content , alpha lp < 0.05). Also, males had greater lean mass and leg lean mass, assessed via DXA (p < 0.05).Anthropometric measures at baseline in males and females are reported in p = 0.09) for an increase in SC content from pre-to-post eccentric damage (p < 0.05) for type I SC content, where males increased in SC content (6.4 ± 0.8 to 11.0 ± 1.7 Pax7+ cells/100 fibers) compared to females (8.5 ± 1.5 to 8.0 ± 1.3 Pax7+ cells/100 fibers), following damage (p < 0.05) in SC content from pre-to-post, but no group differences (p < 0.05) (p = 0.06); males demonstrated a larger increase in total SC (13.0 ± 1.5 to 21.1 ± 3.2 Pax7+ cells/100 fibers), compared to females (14.3 ± 2.1 to 14.9 ± 2.2 Pax7+ cells/100 fibers)  . Additio fibers) .p < 0.05) in SC activation in males (0.7 ± 0.2 to 1.7 ± 0.3 Pax7+/MyoD+ cells/100 fibers) and females (1.2 ± 0.3 to 1.7 ± 0.4 Pax7+/MyoD+ cells/100 fibers) following eccentric damage (p < 0.05) (p = 0.06) in SC activation in males (1.0 ± 0.2 to 1.6 ± 0.3 Pax7+/MyoD+ cells/100 fibers) and females (0.9 ± 0.2 to 1.40 ± 0.3 Pax7+/MyoD+ cells/100 fibers) following eccentric damage (p < 0.05) in total SC activation in males (1.8 ± 0.2 to 3.2 ± 0.5 Pax7+/MyoD+ cells/100 fibers) and females (2.2 ± 0.4 to 3.1 ± 0.6 Pax7+/MyoD+ cells/100 fibers) following eccentric damage (p < 0.05) (p = 0.09) in the proportion of active SC in males (9 ± 2%–20 ± 4%) and females (16 ± 4%–20 ± 5%) (p < 0.05) in the proportion of active SC in males (11 ± 2%–25 ± 6%) and females (14 ± 3%–26 ± 6%) (p < 0.05) but no differences were found between groups  . Likewisc damage . Finally < 0.05) . The pro20 ± 5%)  but no d26 ± 6%) . In totan groups .p > 0.05). Fold changes for mRNA expression are presented with genes of interest normalized to GAPDH. In response to eccentric damage, there was a trend (p = 0.09) for a time × group interaction for Pax7 gene expression, where males (2.1-fold change) had a higher expression, compared to females (1.6-fold change) (p < 0.05) similarly in males  and females   . Both My change) .p > 0.05). There was an increase (p < 0.05) in CXCL8 expression in males (152.2-fold change) and females (105.9-fold change) (p = 0.09) for a time × group interaction for CCL2 expression, where males had a larger (41.1-fold change) increase than females (15.0-fold change) (p < 0.05) in females (12.1-fold change) and showed a trend (p = 0.07) for an increase in males  in males (5.5-fold change) and females (5.8-fold change) to a similar extent  . There w change) . IL6 incr extent .The major finding from this study was that following 300 maximal eccentric contractions, males had a significantly greater increase in SC expansion compared to females. Furthermore, we observed a trend for a greater increase in Pax7 and CCL2 expression in males 48 h following muscle damage compared to females. To our knowledge, this is the first investigation to report a sex-based difference in the SC response, following muscle damage in humans.in vivo, have been conducted in males only. In this study, males experienced a 60% increase (p < 0.05) in total SC content following damage, which is similar to what we have previously reported–in males–using this protocol (p < 0.05 (p = 0.11), nonetheless, this finding is consistent with the greater expansion of SC observed in males. This differential SC response is further corroborated through gene expression analysis, where a trend was detected for greater Pax7 mRNA expression in males, compared to females (Most human studies investigating the SC response, protocol  (Figure protocol . The resprotocol . In our protocol  followin females . TogetheWe sought to determine if there was an altered inflammatory gene expression response between males and females. Since M1-macrophage abundance is elevated at 48 h , we focuThe present observations may reflect underlying sex-based differences in muscle damage after the same relative load of exercise, as has been indicated in previous animal  and humaA notable difference between males and females that could influence muscle repair is the difference in sex hormone profile, specifically a higher concentration of testosterone in males and estrogens in females. While much of testosterone’s effects on muscle inflammation and regeneration have been described , little This study was not without limitations. Firstly, we did not have access to serum samples, and we did not record the menstrual phase during the experimental protocol, thus we were unable to measure or estimate estrogen concentration at the time of the biopsies. However, it is important to recognize that regardless of menstrual cycle phase, females would have had a significantly higher concentration of estrogen compared to males. Second, we were limited to one timepoint following eccentric damage, more timepoints would have permitted a more comprehensive investigation of the early- and late-myogenic and inflammatory responses. Third, we were unable to provide any traditional markers of muscle damage such as creatine kinase serum concentration, a decline in muscle strength, or Z-line streaming due to a lack of tissue availability. Despite these limitations, our results demonstrate that SC expansion is different between males and females following the same relative eccentric-focused bout of muscle damage. Furthermore, the inflammatory response in females, as characterized through gene expression responses was significantly different to that of males. To further reveal sex-based differences in muscle regeneration/repair in humans, future studies should focus on comparing males and females, while accounting for serum estrogen concentration and/or menstrual phase at the time of biopsy. Moreover, an extensive post-damage time-course will allow for more extensive characterization of the inflammatory and regenerative/repair response.