Target compounds were evaluated for their antitubercular efficacy against Mycobacterium tuberculosis H37Rv and antimicrobial activity against five common pathogenic bacterial and three common fungal strains. Three derivatives  displayed significant antitubercular activity with IC50 values of ≤16,760. Compounds derived from trimethoxy substituent scaffolds with monofluoro substitution on the B ring of the chalcone structure exhibited superior inhibition activity compared to corresponding hydroxy analogs. In terms of antimicrobial activity, most compounds  exhibited moderate to potent activity against the bacteria, and the antifungal activities of compounds 3, 13, 15, 20, and 22 were comparable to those of reference drugs ampicillin and fluconazole.Owing to ever-increasing bacterial and fungal drug resistance, we attempted to develop novel antitubercular and antimicrobial agents. For this purpose, we developed some new fluorine-substituted chalcone analogs ( Mycobacterium tuberculosis, is one of the most important diseases worldwide. According to a 2014 report by the WHO, incidences, prevalence, and mortality rates of tuberculosis have globally decreased. However, in 2014 alone, an estimated 9.6 million new cases of tuberculosis and 1.5 million deaths caused by the disease were reportedTuberculosis, caused by The incidences of failure in the treatment of bacterial and fungal infections have increased because of the emergence of multidrug-resistant strains due to misuse of antimicrobial drugsChalcones are compounds with simple chemistry that offer easy synthetic access to various substituted derivatives. As well as being important constituents of various natural products, chalcones, also called α,β-unsaturated ketones, are important synthetic precursors. Chalcones and their synthetic derivatives possess extensive pharmacological properties, such as antihypertensive, antiplatelet, antidiabetic, antineoplastic, antiangiogenic, antiretroviral, anti-inflammatory, antihistaminic, antioxidant, antitubercular, antifungal, anti-invasive, and antiulcer properties,In recent years, compounds containing fluorine have become common as potential lead drugs6In this study, we performed antitubercular and antimicrobial activity studies on eleven B-ring fluoro-substituted chalcones and two nonsubstituted chalcones that we have previously synthesized in an efficient, high-yielding manner1H- and 13C-NMR Spectra: Varian 400 and Bruker 400 spectrometers. Elemental analysis results were obtained on a Leco CHNS-932 instrument.All reagents used were commercially available unless otherwise specified and all solvents were distilled before use. Melting points were measured with Gallenkamp melting point devices. IR Spectra: PerkinElmer Spectrum One FT-IR spectrometer. E)-3-(4-Fluorophenyl)-1-prop-2-en-1-one (11);(1)  in MeOH (20 mL) 4-fluorobenzaldhyde (7) (0.6 mL 7.6 mmol) and 50% KOH solution (10 mL) was added sequentially and stirred for 15 h at room temperature. After 15 h, solvent was evaporated; 2 M HCl solution (15 mL) was added and crude product was extracted with DCM (3 × 20 mL). The combined extracts were dried over Na2SO4. The solvent was removed in vacuo and the remaining residue purified via coloumn chromatography over silica gel using gradient elution with EtOAc and Hexanes to yield compound 11, as a yellow solid (80% yield). Rf (EtOAc/Hexanes 30:70) = 0.27; MP = 122–123 °C; IR  vmax 3502, 2941, 2841, 1651, 1599; Anal. calcd for C18H18O4: C, 68.35; H, 5.42; Found: C, 68.16; H, 5.38.To a solution of 2,4,6-trimethoxyacetophenon  δ 7.52–7.48 , 7.32 , 7.07–7.01 , 6.87 , 6.15 , 3.84 , 3.76 .13C NMR  δ 194.1, 164.0 , 162.7, 159.1, 142.8, 131.5, 130.4 , 129.0, 116.1 , 111.9, 90.9, 56.1, 55.7.3, 4, 9–15, 20–23 were obtained by agar dilution in duplicate as recommended by the Clinical Laboratory Standards Institute (CLSI)12,M. tuberculosis H37Rv was used as the standard strain and was provided by Refik Saydam National Public Health Agency, National Tuberculosis Reference Laboratory, Ankara, Turkey. Stock solutions of the tested compounds and reference compounds were prepared in DMSO/H2O (50%) at a concentration of 1000 µg/mL. These solutions were then filtered through a 0.22-µm membrane filter . Middlebrook 7H10 agar medium  was supplemented with oleic acid–albumin–dextrose–catalase . Compounds and control agents were added to obtain an appropriate final concentration in the medium. The final concentrations of INH and EMB were 0.2–1 µg/mL and 5–10 µg/mL, respectively. Compounds were prepared at final concentrations of 5, 10, 20, 40, and 80 µg/mL. Agar without any reference or tested compounds was used as a positive growth control, and 3.0 mL of prepared medium was dispensed into sterile tubes. The DMSO concentration in the final solutions was not above 1% for antimycobacterial activity.Agar proportion method: The MIC values of each synthesized compound –2 to 10–4 dilutions were obtained from McFarland standard No. 1. Then, 0.1 mL of the diluted suspension was inoculated onto the control and the other tubes with compounds in different concentrations. The tubes were incubated at 37 °C in a 5% CO2 atmosphere for 3 weeks. The MIC values were defined as the lowest concentration that inhibited more than 90% of the bacterial growth, and the results for INH and EMB were interpreted according to the CLSI. The MIC was considered as the lowest concentration that showed no visible colonies in all dilutions. The biological activity studies of the compounds were performed twice. The results of these two studies were almost the same. The IC50 values were calculated as the average activity values for the compounds. The results are given in Inoculum preparation: H37Rv was maintained in Lowenstein–Jensen medium. A culture suspension was prepared by subculturing in Middlebrook 7H9 broth  supplemented with 10% OADC at 37 °C for 7–10 days, until a density corresponding to 10,Staphylococcus aureus (ATCC 25925), Streptococcus pyogenes (ATCC 19615), Enterococcus faecalis (ATTC 29212), Escherichia coli (ATCC 25293), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (ATCC 10231), Candida glabrata (RSHM 40199), and Candida parapsilosis (ATTC 22019).Antimicrobial susceptibility testing was performed by the modification of literature methods14The fungal and bacterial cell inoculum were prepared from a stock culture grown in tryptic soy agar (TSA) at 28 °C for 24 h, and Mueller–Hinton agar (MHA) at 37 °C for 24 h, respectively. The microorganism suspension concentrations were adjusted according to McFarland 0.5 turbidity tubes using sterilized saline. Stock solutions of the title compounds were prepared in DMSO at 1000 µg/mL. A modified microdilution test was applied for antimicrobial activity, and the experiments were run in duplicate independently.For antifungal activity testing, 100 µL Tryptic Soy Broth (TSB) was added to each of the 11 wells. A 100 µL aliquot of the tested chemical solution was added to the first well, and twofold dilutions were prepared. Then, 5 µL of fungal suspension was added to each tube except the last one, which acted as the control well.For antibacterial activity testing, 100 µL Mueller–Hinton broth (MHB) was added to each of the 11 wells. A 100 µL aliquot of the chemical derivative solution was added to the first tube, and twofold dilutions were prepared. Then, 5 µL of the bacterial suspension was added to each tube, except the last control well. A control tube containing 5 µL of the fungal and bacterial suspensions alone without the tested compounds was also prepared. All plates were incubated at 28 °C (for fungi) and at 37 °C (for bacteria) for 24 h. After incubation, the MICs  and 3 weWe previously reported the synthesis of 11 B-ring fluoro-substituted and two nonsubstituted chalcone derivatives3, 9–11, and 12 in yields of 80–95%. Hydrogenation of the chalcone compounds over Pd–C gave 4, 13, 14, and 15 in yields of 70–90%. Trihydroxy-chalcones were synthesized using the same procedure. Before the condensation reaction, the methoxymethyl ether (MOM) group was used to protect the hydroxyl groups. The base-catalyzed reaction was then used to afford MOM-protected chalcones. After deprotection, hydrogenation of 20 and 21 over Pd–C gave the target compounds 22 and 23 in yields of 80% and 85%, respectively. The general method for the synthesis of these chalcones is briefly described in Using this procedure11 were previously synthesized by our group11 and its structural details.All compounds except 3 (the methoxy derivative with non fluoro substituent) for activity modulation was made through preparation of compounds with fluoro substituents on the B ring only , and with hydroxyl substituents on the A ring and fluoro substituents on the B ring (20 and 21). Versions of these compounds with semisaturated or saturated linkers were also prepared . The synthesized compounds were fully characterized with common spectroscopy techniques.Modification of compound 3 was evaluated for its antimicrobial efficacy against the Mycobacterium tuberculosis H37Rv strain and several other bacteria and fungi. The different functional groups carry on aromatic rings of chalcones are responsible for their antimicrobial activities. The details of the substitution patterns in the target compounds are presented in To evaluate the potential of the basic pharmacophore of chalcone, 3, 4, 9–15, and 23) were screened against M. tuberculosis H37Rv using the agar proportion method. Isoniazid (INH) and ethambutol (EMB) were used as the positive drug standards for the assay. The in vitro antimycobacterial activity in terms of the minimum inhibitory concentration (MIC) values of the target compounds and the standard drugs are shown in 3, 9, and 10 show significant activities, with IC50 values ≤16,760, and compounds 11 and 12 exhibit moderate activities with IC50 values ≤31.63. The remaining eight compounds 4, 13–15, 20–22, and 23 exhibit very low activity. The compounds derived from trimethoxy and non- or monofluoro-substituted chalcone scaffolds exhibit superior inhibition activity to their hydroxyl- and difluoro-substituted analogs. This observation clearly indicates that the trimethoxy A ring and fluoro groups on the B ring enhance the inhibition activity of the molecules. Furthermore, these results suggest that further structural modification of the above-mentioned drug molecules  using a structure–activity relationship (SAR) approach could be a promising strategy for the identification of new leads toward the development of potent antitubercular agents.All the target compounds  against the five common pathogenic bacterial strains S. aureus, S. pyogenes, E. faecalis, E. coli, and P. aeruginosa is shown in The antibacterial activities of the target compounds  exhibit moderated inhibition with MICs of 62.5 and 125 µg/mL, as compared to the standard fluconazole (31.25 µg/mL).Compounds 13, 15, and 21 exhibit the highest activity against P. parapsilosis, with MICs of 31.25 µg/mL, whereas all other compounds exhibit low inhibitory activity compared to that of fluconazole, with MICs of 62.5 µg/mL. Compound 21, the hydroxyl and difluoro-substituted chalcone analog, exhibits the highest activity.Compounds 3, 4, 9–15, and 20–23 was explored using the data presented in The SAR of compounds p on the antifungal activity of the compounds appears to be important: as Log p increases, the antifungal activity increases. Furthermore, the use of semi-saturated linker compounds appears to improve activity against all targets.More specifically, compounds with fluoro substituents in position 2 and/or position 5 of the B ring in the chalcone structure exhibit significantly increased potency against the microbial strains. The influence of Log Glycyrrhiza inﬂata, and 24 and 25, present in Piper sanctum, have been reported to exhibit potent antitubercular activity against Mycobacterium tuberculosis, with MICs of 7.1, 32, and 4.0 µg/mL, respectively  is represented in via two steps. The compounds tested for antitubercular activity were divided into two series; series A comprised trimethoxy-substituted compounds 3, 4, 9–15 derived from the parent structure 3 with fluoro substituents in position 2–5 of the B ring, and series B contained three hydroxyl substituents on the A ring and a mono- or difluoro-substituted B ring (20–23).The synthetic route for chalcone derivatives  exhibited the greatest antitubercular activity with IC50 values ≤16,760, which are comparable to the antitubercular drugs INH and EMB used as controls.Preliminary studies revealed that compounds (3 for activity modulation was made by introducing fluorine atoms or saturation. These compounds were found to be either far less active than 3 or inactive. It is widely known that molecular ﬂexibility plays an important role in drug–protein interactions. However, we found that the conformationally restricted chalcones were more active than the other chalcones. This modification was made in such a way that the total lipophilic nature of the compounds was similar (The Log p values for compounds are shown in Modification of compound in vitro antimicrobial activity against several bacterial and fungal strains. The tested compounds were inactive against bacteria and fungi, and they did not exhibit significant antibacterial and antifungal activities, except against S. aureus, in comparison to ampicillin and fluconazole. However, the chalcone analogs exhibited considerable antimicrobial activity. Interestingly, compounds 3 and 4 exhibited wider activity than the other compounds. Compounds 3, 20, and 11, 23 exhibited higher antibacterial and antifungal activity against S. aureus and C. albicans, respectively.The synthesized compounds were also evaluated for their In conclusion, assessment of the biological activity of these molecules indicated that conformationally restricted chalcones are superior to their straight chain analogs, possibly due to molecular ﬂexibility, and may be good leads for the future development of antitubercular drugs.


Locus Aml showed differential banding patterns in males and females similar to those seen for humans and correctly assigned sexes of known identity. Altogether, 58 different alleles were found with an average 3.05 ± 0.28 alleles per locus. Mean observed (Ho), and expected heterozygosity (He) were 0.93 ± 0.03 and 0.52 ± 0.05, respectively.Of 21 STRs in PowerPlex A few studies have provided direct evidence for an association between genetic variability and reproductive performance in natural populations , 2. Howe® 21 System  that cross-amplified in a single multiplex reaction.In this study, we identified 19 loci from the PowerPlexGeneSeek (http://www.neogen.com/geneseek). Genomic DNA was extracted from hair follicles using Qiagen DNeasy Blood and Tissue kit  following the manufacturer’s instructions. All DNA extraction procedures were done in aseptic conditions.Hair samples from three Chimpanzees  were provided by the Zoo Officials in India. The samples were collected on commercially available hair sample collection cards, ® 21 System, Promega, USA) designed for humans was used to amplify 21 loci in chimpanzees. The PowerPlex® 21 System is used for human identification applications including forensic analysis, relationship testing and research use. The system allows co-amplification and four-color fluorescent detection of 21 loci (20 STR loci and Amelogenin), including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, Amelogenin (henceforth Aml), CSF1PO, FGA, Penta D, Penta E, TH01, TPOX and vWA [® 21 5X Master Mix, PowerPlex® 21 5X Primer Pair Mix and sterile water, amplification grade, following manufacturer’s instructions . We also included positive and negative control reactions to monitor PCR contamination. All the amplification reaction were performed on GeneAmp 9700 thermocycler  in a total volume of 25 µl. Thermal cycling condition was followed as per the PowerPlex® 21 System . The amplified PCR products were subjected for the fragment analysis on the ABI 3130 Genetic Analyzer .A commercial kit . After scoring and re-arrangement of multi-locus genotype data was processed further for the assessment of genetic diversity estimates. Estimates of genetic diversity, including observed number of alleles per locus (Na), effective number alleles (Ne), inbreeding coefficient (F), observed (Ho) and expected heterozygosity (He), were obtained using using GENEALEX version 6.41 software .® 21 System , two loci (D5S818 and D12S391) failed to amplify across the chimpanzee samples and 19 loci were found to be polymorphic. Locus Aml, a non-STR marker, demonstrates differential banding patterns in males and females humans [® 21 System has discriminating power in correctly assigning sexes in Chimpanzees, hence an extended developmental validation of the PowerPlex® 21 System.Out of 21 STRs in the PowerPlexHo), and expected heterozygosity (He) were 0.93 ± 0.03 and 0.52 ± 0.05, respectively. The mean unbiased expected heterozygosity (UHe) was 0.63 ± 0.06.. The fixation index (F), a representation of inbreeding was in negative, i.e., − 0.89 ± 0.17, representing an outbred population. Observed profiles of seven loci,  exhibited to be relatively more heterozygous and showed numerous alleles, i.e. ≥ four alleles/locus, indicating a strong signature that these three animals are genetically unrelated and might have come from the different genetic tracts.Altogether, 58 different alleles were found across 19 loci. The number of observed alleles ranged from 5  to 1 (TH01), with an overall mean number of alleles per locus of 3.05 ± 0.28. The observed number of alleles for all loci exceeded the effective number of alleles, with mean observed 3.05 ± 0.28  in chimpanzee, extending developmental validation of the PowerPlex® 21 System for individual identification and molecular sexing of chimpanzees. The results showed high genetic variability in the analyzed individuals and three chimps were genetically distinct and with no evidence of inbreeding.This study has been the first attempt to establish human identification STR kit- PowerPlexThis system can be used for population estimation following capture-mark-recapture methods with larger sample sizes, assigning population genetic structure in wild populations, demographic history, and investigating gene flow and other associated parameters of Chimps in wild and captivity.ID) and probability of identity for siblings (PSIB) that can be used for individual identification through mark-recapture analysis.This study identified heterologous microsatellites for Chimps and include as such no potential limitations. However, if we had more number of chimpanzee samples (reference and pellets), we could certainly propose the combination of microsatellites based on probability of identity (P


Skin reactions and cardiotoxicity are one of the most common side effects of doxorubicin in cancer patients. The main mechanisms based on the etiopathogenesis of these reactions are mediated by the overproduction of proinflammatory cytokines, metalloproteases, and the disruption of mitochondrial homeostasis. Ozone therapy demonstrated anti-inflammatory effects in several preclinical and clinical studies. The aim of this research is based on the evaluation of cardioprotective and dermatoprotective effects of ozone during incubation with doxorubicin, giving preliminary evidences for further studies in the field of cardio-oncology. μg/mL. Cell viability and multiple anti-inflammatory studies were performed in both cell lines, with particular attention on the quantification of interleukins, leukotriene B4, NF-κB, and Nrf2 expressions during treatments.  Human skin fibroblast cells and human fetal cardiomyocytes were exposed to doxorubicin at subclinical concentration (100 nM) alone or combined with ozone concentrated from 10 up to 50 μg/mL with a plateau phase at higher concentration. Ozone also demonstrated anti-inflammatory effects decreasing significantly the interleukins and proinflammatory mediators in both cells.  Ozone decreased significantly the cytotoxicity of doxorubicin in skin fibroblasts and cardiomyocytes after 24 h of incubation. The best cytoprotective effect of ozone was reached to 30  Ozone exerts cardioprotective and dermatoprotective effects during incubation with doxorubicin, and the involved mechanisms are mediated by its anti-inflammatory effects. The overall picture described herein is a pilot study for preclinical studies in oncology. The ozone therapist uses conventionally ozone as a tool to disinfect hospital instruments and tissues, as well as to reduce the general pain and inflammation  method according to the instructions provided by Dojindo Molecular Technologies Inc. Cardiomyocytes were cultured in a Claycomb medium  containing 10% fetal bovine serum, 100 U/mL penicillin/streptomycin, 0.1 mM norepinephrine, and 2 mM L-glutamine and maintained at 37°C in 5% COterature , 14, alo74  were quantified through a Tecan Infinite M200 plate reader with the associate I-control software. The MMP of fibroblasts and cardiomyocytes was calculated as the red/green fluorescence ratio.MitoProbe™ JC-1 Assay Kit (Thermo Fisher kit) was used for the determination of the mitochondrial matrix potential (MMP) in skin fibroblasts and cardiomyocytes, according to the manufacturer's protocol. Following treatment with doxorubicin at subclinical concentration (100 nM) and ozone at 10, 20, 30, 40, and 50 κB transcription factor assay kit , according to the manufacturer's recommendations. NF-κB complexes were captured by binding to a consensus 5′-GGGACTTTCC-3′ oligonucleotide immobilized on a 96-well plate. Bound NF-κB was quantified by incubating with anti-p65 primary antibody followed by horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG and spectrophotometric detection at a wavelength of 450 nm using a microplate spectrofluorometer . Data were expressed as the percentage of NF-κB/DNA binding relative to control cells.Fibroblasts and cardiomyocytes were exposed to doxorubicin at 100 nM alone or in combination with ozone at different concentrations for 6 h, and then nuclear extracts were analyzed using the TransAM NF-μg/mL), we performed a nuclear extraction in both cell lines, and 10 μg of the extracts were incubated in a 96-well plate that was coated with oligonucleotides containing a consensus binding site for Nrf2. After 1 h of incubation, we washed the plates three times, slowly, with cold PBS and incubated with 100 μl of a 1 : 1000 dilution of polyclonal antibody against Nrf2. After the appropriate washing with cold PBS, we incubated the plates with 100 μl of a 1 : 1000 dilution of horseradish peroxidase-conjugated, anti-secondary antibody at 25°C. Then, the fold expression of Nrf2 was obtained dividing the absorbance of treated cells and untreated (control) cells at a wavelength of 450 nm using a microplate spectrofluorometer .For the evaluation of Nrf2 activity during incubation with doxorubicin (100 nM) and ozone (from 10 to 50 μM) as the positive control because it is the main precursor of leukotrienes, doxorubicin (100 nM) alone or coincubated with ozone at 10, 20, 30, 40, and 50 μg/mL. During the incubation period, cells were also exposed to 5 μM calcium ionophore A23187, 1.6 mM CaCl2 at 37°C for 4 h. Then, we performed the appropriate ELISA method for the quantification of leukotriene type B4, expressed as pg/mL  following the supplier's instructions [Leukotriene B4 expression in cardiomyocytes and skin fibroblasts was determined through the following method: both cells were incubated with arachidonic acid (10 ructions .μg/mL) for 5 h before exposure to doxorubicin always at subclinical concentration (100 nM) for 12 h. As performed for the other experiments, it is crucial to say that we decided to use this concentration of anticancer drug for simulating at cellular level the main clinical exposure of organs (like the heart) to doxorubicin after intravenous administration in humans [We quantified the expression of proinflammatory cytokines and metalloproteases involved in the skin and cardiac toxicities induced by chemotherapies through the ELISA methods, as described in detail in other works , 19. Aftn humans . After tp < 0.05 is considered as statistically significant.The results reported are the average of three independent experiments ± SD (standard deviation). We performed a one-way analysis of variance (ANOVA) and by a subsequent Tukey's multiple comparison test in SigmaPlot Software for the determination of the difference between the groups. For statistical analysis of all data, μM, in agreement with our previous research [μM at the higher ozone concentration (50 μg/mL). Coincubation of cardiomyocytes with doxorubicin 20 μM and ozone at 10, 20, 30, 40, and 50 μg/mL increases their viability 1.8, 2.5, 3.3, 3.6, and 4.2 times, respectively, compared to only doxorubicin-treated cells . Coincubation of cardiomyocytes with doxorubicin 50 μM and ozone at 10, 20, 30, 40, and 50 μg/mL increases their viability 2.1, 4.5, 5.5, 6.4, and 7.3 times, respectively, compared to only doxorubicin-treated cells . A similar but lower significative behaviour was seen in skin fibroblasts with an overall cytoprotective effects of 10–15% of ozone at higher concentration coincubated with doxorubicin. As an example, coincubation of skin fibroblasts with doxorubicin 50 μM and ozone at 10, 20, 30, 40, and 50 μg/mL increases their viability 1.3, 1.6, 2.1, 2.3, and 2.4 times, respectively, compared to only doxorubicin-treated cells .As shown in research  in cardip < 0.001 for both). Coincubation with ozone leads to an improvement of mitochondrial matrix potential in cardiomyocytes 10.2, 28, 36, 43.2, and 46.1% compared to only doxorubicin-treated cells . A similar behaviour was seen in skin fibroblasts, where the coincubation of ozone with doxorubicin improves the mitochondrial matrix potential in cardiomyocytes 10.2 (not significant), 21.1, 29.4, and 32% compared to only doxorubicin-treated cells   .κB plays a crucial role in cardiovascular diseases, cardiotoxicity, and skin adverse effects of several anticancer drugs. To investigate whether ozone affects NF-κB activation, which is critical for transcriptional activity, the DNA binding activity of NF-κB was analyzed by ELISA. As shown in κB in cardiomyocytes and skin fibroblasts, respectively (p < 0.001 for both) compared to untreated cells. Notably, the coincubation of doxorubicin and ozone leads to a concentration-dependent reduction of p65/NF-κB expression with a plateau phase at 40 and 50 μg/mL. In cardiomyocytes . In skin fibroblasts . Increasing ozone concentration over 40 μg/mL did not show more anti-inflammatory activity related to NF-κB activity, indicating a plateau phase.The activation of the key proinflammatory mediator NF-myocytes , left, oroblasts , right, p < 0.001 for both). Incubation with doxorubicin increases around 2.8 times the leukotriene B4 production compared to untreated cells (p < 0.001 for both). Notably, in cardiomyocytes . In skin fibroblasts . Also in this case, ozone at 40 and 50 μg/mL did not show any greater anti-inflammatory effects both in cardiomyocytes and skin fibroblasts.Leukotrienes are the progenitors of the inflammation related to the activation of prostaglandins . The infmyocytes , left ozroblasts , right, μg/mL increased the Nrf2 expression 1.2, 1.4, and 1.5 times, respectively, compared to only doxorubicin-treated cells . In skin fibroblasts, we have seen the same behaviour (Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a significant role in regulating the expression of antioxidant and cytoprotective enzymes in response to oxidative stress . Herein,ehaviour , right, α, MMP-2, and MMP-9 3.2, 2.5, 3, 2.8, 2.3, and 2 times, respectively, compared to untreated cells. Ozone at 10, 20, and 30 μg/mL decreased 10–13, 21–25, and 23–28% the production of all analyzed cytokines and growth factors compared to only doxorubicin-exposed cells . Increasing ozone concentrations seems to not decrease significantly the production of these cytokines indicating a plateau phase, corroborating biological data described in previous paragraphs. Moreover, in skin fibroblasts, doxorubicin at 100 nM increased the production of IL-1, IL-8, IL-6, TNF-α, MMP-2, MMP-9, 2, 2.3, 2.2, 2.4, 2, and 2.1 times, respectively, compared to untreated cells. Ozone at 10, 20, and 30 μg/mL decreased 8–12, 18–21, and 20–22% the production of these cytokines and growth factors compared to only doxorubicin-exposed cells . Also in this case, greater doses of ozone do not appear to have a greater anti-inflammatory effect thus having a pattern similar to that observed in cardiomyocytes (We previously demonstrated anti-inflammatory and anticancer properties of ozone in the cellular model of colon  and melamyocytes .β plays a major role in the genesis of chronic inflammation. Ozone is an innovative method for decreasing the inflammation status in preclinical and clinical experiences. Our research group recently demonstrated the abilities of ozone in increasing 5-fluorouracile and cisplatin cytotoxicity and decreasing interleukin secretion in human colon cancer cells [Doxorubicin is a cytotoxic drug used from several years in oncology as therapy for many diseases like leukemias and lymphomas, as well as breast, lung, and other solid cancers . Howeverer cells . Herein,er cells . The reser cells . In conc


Palmitic acid (PA) is the most common saturated long-chain fatty acid that causes damage to heart muscle cells. However, the molecular mechanism of PA toxicity in myocardial cells is not fully understood. In the present study, we explored the effects of PA on proliferation and apoptosis of H9c2 cardiomyocytes, and uncovered the signaling pathways involved in PA toxicity. Our study revealed induction of both oxidative and endoplasmic reticulum (ER) stresses and exacerbation of apoptosis in PA-treated H9c2 cells. Inhibition of oxidative stress by N-acetylcysteine (NAC) reduced apoptosis and decreased ER stress in PA-treated H9c2 cells. Moreover, inhibition of ER stress by 4-phenyl butyric acid decreased apoptosis and attenuated oxidative stress. In summary, the present study demonstrated that oxidative stress coordinates with ER stress to play important roles in PA-induced H9c2 cell apoptosis. Diets rich in high fat foods, especially saturated fats, cause obesity, leading to serious health problems, such as type 2 diabetes and lipotoxic cardiomyopathy . It has The ER is an indispensable and elaborate eukaryotic organelle that is primarily responsible for synthesis, packaging, and assembly of secretory and membrane proteins . Any phyOxidative stress plays a critical role in the pathogenesis of diabetic cardiomyopathy, which might impair antioxidant defense systems . ReactivIn the present study, PA-treated H9c2 cells were used as a model to examine the molecular mechanism of lipotoxic cardiomyopathy. Here, we investigated whether oxidative and ER stress pathways are involved in H9c2 cell apoptosis and explored the relationship between oxidative and ER stresses in PA-induced H9c2 cell apoptosis.N-acetylcysteine , PA, 4-phenylbutyrate , and bovine serum albumin (BSA) were purchased from Sigma–Aldrich . Fetal bovine serum (FBS) was purchased from Gibco . Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone . H9c2 cardiomyocytes were purchased from the Chinese Academy of Sciences . A caspase 3 Activity Assay Kit was purchased from the NanJing JianCheng Bioengineering Institute . NaOH, phosphate-buffered saline (PBS), penicillin, and gentamicin were obtained from Solarbio . A CCK8 Assay Kit, ROS Assay Kit, and Annexin V-PE/PI Apoptosis Analysis Kit were purchased from the Beyotime Institute of Biotechnology . TRIzol and a PrimeScript® RT Reagent Kit were purchased from Invitrogen . PVDF membranes were obtained from Millipore .PA was prepared by soaping palmitate with sodium hydroxide and mixing with BSA. PA (20 mM in 0.01 M NaOH) was incubated at 70°C for 30 min. Then, the fatty acid soaps were complexed with 5% fatty acid-free BSA in PBS in a 1:3 volume ratio to produce a 5 mM PA stock solution, which was stored at −20°C. Before application, the stock solution was diluted in culture medium.2. The growth medium was changed every 3 days. When the cells reached 70–80% confluence, they were treated with 100–800 μM PA for 24 h. In other experiments, H9c2 cells were exposed to 400 µM PA in the presence or absence of 500 nM 4-PBA and 2 mM NAC. After incubation for 24 h, the cells were collected to assess cell viability, apoptosis, caspase 3 activity, and B-cell lymphoma 2 (BCL-2)-associated X protein (BAX), GRP78, CHOP, and NOX2 expression.H9c2 cells were cultured in DMEM supplemented with 10% FBS, 50 μg/ml penicillin, and 50 μg/ml gentamicin in a humidified incubator at 37°C with 5% CO4 per well in 96-well plates. After treatment, 10 μl CCK8 was added to each well, and the cells were incubated for 2 h at 37°C. The number of viable cells was measured by a microplate reader  at 450 nm.H9c2 cell proliferation was monitored by a CCK8 assay, in accordance with the manufacturer’s instructions. Cells were plated at 2 × 10Apoptosis was determined using the Annexin V-PE/PI Apoptosis Analysis Kit. After treatments, the cells were collected and resuspended in 500 μl binding buffer. After incubation with 10 μl Annexin V-PE and 5 μl PI for 15 min at 25°C in the dark, the apoptotic rates of the cells were detected by a flow cytometer . Early apoptotic cells were Annexin V+/PI− cells, late apoptotic cells were Annexin V+/PI+ cells, necrotic cells were Annexin V−/PI+ cells, and Annexin V−/PI− cells were considered as live cells.ROS measurement was carried out in accordance with the procedure of the ROS Assay Kit. After treatments, the cells were incubated with 10 µM DCFH-DA at 37°C for 20 min. Then, the cells were washed three times, and staining with the ROS-sensitive dye was assessed under an inverted fluorescence microscope  at an excitation wavelength of 488 nm and emission wavelength of 525 nm. Fluorescence was examined by image analysis software ImageJ (Version 1.49).Caspase 3 activity was measured using a caspase 3 Activity Colorimetric Assay Kit. After treatments, cells were harvested by scraping, collected by centrifugation, and incubated in lysis buffer on ice for 15 min. Then, the lysate was centrifuged at 15000 rpm for 15 min at 4°C, and the protein content was determined, after which the caspase 3 substrate was measured using the microplate reader at 405 nm.After treatments, cells were collected and lysed with TRIzol reagent. Total RNA was reverse transcribed using the PrimeScript® RT Reagent Kit, in accordance with the manufacturer’s instructions. The qRT-PCR procedure was carried out using a Bio-Rad IQ5 and Bio-Rad IQ5 Optical System Software (Bio-Rad). The PCR cycling conditions were 1 cycle of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. Primers are listed in Supplementary Table S1. β-Actin served as a reference gene. mRNA expression of oxidative stress and ER stress markers after the treatments is shown in Supplementary Figures S1 and S2.After treatments, cells were collected, washed with ice-cold PBS, and lysed with RIPA buffer. The total protein concentration was then measured by the BCA assay. Total protein (50 µg) in each sample was loaded into each well of a 12% SDS/PAGE gel and separated by electrophoresis. Proteins were then transferred on to PVDF membranes. After blocking in TBST with 10% dry nonfat milk for 2 h, the membranes were incubated with a primary antibody (Supplementary Table S2) overnight at 4°C. After washing, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase at 37°C for 30 min. Immunoreactive bands were visualized using a Super Signal West Pico kit, in accordance with the manufacturer’s instructions. Protein band densities were semi-quantitated by densitometric analysis using ImageJ (Version 1.49).t test with SPSS software, version 13.0 .All experiments were repeated at least three times for each group. Data are presented as the mean  ±  S.E.M. Results were analyzed by ANOVA, followed by Fisher’s least significant difference test and the independent samples Student’s 50 value was approximately 400 µM (P=0.0032). In addition, PA induced ROS generation  was measured by a colorimetric assay and Western blotting. PA enhanced the levels of caspase 3 activity in a dose-dependent manner from 200 to 800 µM concentrations compared with the control group at 24 h A. In addTo investigate whether ER stress is involved in PA-induced apoptosis, we determined the expression of ER stress marker proteins GRP78 and CHOP. Our results showed that ER stress was active and expression of CHOP and GRP78 was up-regulated at mRNA and proteins levels in a dose-dependent manner from 200 to 800 µM concentrations compared with the control group at 24 h (P=0.2678) and partially reversed the expression of NOX2 caused by PA  induced by PA C,D and eIn the present study, we examined the mechanism of saturated fatty acid-induced cardiomyocyte injury. To this end, we used PA to investigate the mechanisms of lipotoxicity in H9c2 cells. We first evaluated the effect of PA on the proliferation of H9c2 cells. Our data showed that PA reduced cell viability, supporting previous studies showing that PA inhibits cell proliferation and induces apoptosis in many cell types and cell lines, such as neuronal cells , stem ceOxidative stress plays a major role in the pathogenesis of diabetic cardiomyopathy . StudiesNext, we detected expression of apoptosis-related genes, including BAX and caspase 3, during PA-induced H9c2 cell apoptosis. Caspase 3 is processed into cleaved caspase 3 in the early steps of apoptosis, and expression of caspase 3 positively correlates with the rate of apoptosis , suggestin vitro and in vivo [ER stress is a cellular rescue mechanism that is activated to ease stress during various pathophysiological insults. However, continuous and excess ER stress pathway activation can result in apoptosis . Previou in vivo . Taken tTo understand the role of oxidative stress in PA-mediated H9c2 cell apoptosis, we suppressed oxidative stress by NAC treatment. The results showed that NAC dramatically decreased the concentrations of ROS. Moreover, GRP78 and CHOP were significantly decreased after NAC treatment. In addition, NAC partially reversed PA-induced cell apoptosis and the decrease in cell viability. These results were consistent with previous studies showing that PA induces oxidative stress and apoptosis in pancreatic β-cells . Our stuTo analyze the role of ER stress in PA-mediated H9c2 cell apoptosis, we suppressed ER stress by 4-PBA treatment. We found that inhibition of ER stress by 4-PBA obviously rescued PA-triggered cell apoptosis, the decrease in cell viability, and expression of GRP78 and CHOP in H9c2 cells. Our data further support a previous study showing that PA induces apoptosis in primary cardiomyocytes via ER stress . This isPrevious studies have shown the association of oxidative and ER stresses with apoptosis ,16. Our Next, we investigated the effects of ER stress on ROS generation. Inhibition of ER stress by 4-PBA significantly decreased ROS generation and NOX2 expression. These results suggested that ER stress is one of the causes of oxidative stress in PA-induced H9c2 cell apoptosis. Moreover, blocking oxidative stress by NAC decreased ER stress, suggesting that ROS generation was an upstream factor in PA-induced H9c2 cell apoptosis. Conversely, blocking ER stress with 4-PBA significantly decreased oxidative stress. These results indicated that oxidative and ER stresses interact with each other during PA-induced cell apoptosis. The possible mechanism may be that oxidative stress disrupts ER homeostasis and causes ER stress. Therefore, inhibition of oxidative stress suppresses ER stress during PA treatment. In addition, persistent ER stress may cause mitochondrial dysfunction that further induces oxidative stress. Thus, inhibition of ER stress can also inhibit oxidative stress. However, the exact underlying mechanism requires further investigation.In summary, our study demonstrates that both oxidative and ER stresses are involved in PA-induced H9c2 cell apoptosis, and there is a cross-talk between oxidative and ER stresses during this process. The present study offers new insights into the molecular mechanisms of lipotoxicity in diabetic cardiomyopathy.


Rhamnus davurica Pall. (R. davurica) has been used as a traditional medicinal herb for many years in China and abroad. It has been well documented as a rich source of flavonoids with diversified structures, which in turn results in far-ranging biological activities, such as anti-inflammation, anticancer, antibacterial and antioxidant activities. In order to further correlate their anticancer potentials with the phytochemical components, the fingerprint profile of R. davurica herb from Dongbei was firstly investigated using HPLC-ESI-MS/MS. Thirty two peaks were detected and identified, 14 of which were found in R. davurica for the first time in this work. Furthermore, a total of 23 peaks were resolved as flavonoids, which are the major components found in R. davurica. Meanwhile, the antiproliferative activities against human cancer cells of HT-29 and SGC-7901 in vitro exhibited distinct inhibitory effects with IC50 values at 24.96 ± 0.74 and 89.53 ± 4.11 μg/mL, respectively. Finally, the general toxicity against L-O2 cells displayed a much higher IC50 at 229.19 ± 8.52 μg/mL, which suggested very low or no toxicity on hepatic cell viability. The current study revealed for the first time the correlations between the flavonoids of R. davurica with their antiproliferative activities, which indicated that the fingerprint profile of flavonoids and their anticancer activities could provide valuable information on the quality control for herbal medicines and their derived natural remedies from this valuable medicinal plant. Flavonoids are one of the most ubiquitous polyphenolic secondary metabolites existing in natural products, which are widely found in plants and their corresponding derived foods. In plants, the basic chemical skeleton of flavonoids is biosynthesized by a series of condensation reactions between three malonyl residues (A ring) and hydroxycinnamic acid (B ring), namely a C6-C3-C6 skeleton . These sRhamnus (Rhamnaceae) is known to be rich with phenolic substances, such as flavonoids, anthraquinones and naphthalenes − ion at m/z 303 and further suffered neutral losses of 18 Da (H2O) and 28 Da (CO) of the C ring to produce the fragment ions of the [M − H − H2O]− ion at m/z 285 and the [M − H − CO]− ion at m/z 275. Typically, the ions at m/z 151 and m/z 125 correspond to the characteristic retro-Diels–Alder (RDA) cleavage between different chemical bonds of the C ring. In addition, fragment ions of [M − H − H2O − 44]− at m/z 241, [M − H − CO − 58]− at m/z 217 and [M − H − CO − 58 − 18]− at m/z 199 were probably produced by the successive neutral loss of the CO moiety, the CO + CH2O moiety and the H2O moiety. Besides, the fragment ion at m/z 175 was regarded as the neutral loss of the B ring moiety from [M − H − H2O]− at m/z 285. Based on the comparisons of the proposed fragmentation pathways and the MS/MS spectra in the previous study − at m/z 287) shared the same basic parent skeleton of dihydroflavonol with Peak 9 and was identified as aromadendrin  due to the absence of hydroxyl group (-OH) at the C-3′ position of the B ring − at m/z 285 in the negative mode, with several fragment ions, such as [M − H − CH3]− at m/z 270, [M − H − CO − C]− at m/z 243 and [M − H − C8H8O]− at m/z 165. Additionally, the ion at m/z 119 corresponded to the characteristic RDA-cleavage of the C ring and further to the dissociation of the acetenyl moiety (m/z 93). Based on the comparison of the proposed fragmentation pathways and the MS/MS spectra of the previous study, Peak 29 was suggested as sakuranetin [Peak 27 showed the [M − H]− ion at m/z 325 and its fragment ions at m/z 271, 185 and 125, which coincided with Peak 24 and presumably was identified as a naringenin derivative. For Peak 28 ([M − H]− at m/z 551), those MS/MS fragment ions at m/z 285, 165 and 119 were identical to those of Peak 29. Tentatively, the precursor ion at m/z 551 was formed by two sakuranetin with the further neutral losses of H2O and H2. Hence, Peak 28 was determined to be the sakuranetin dimer, which was found in R. davurica for the first time in this study. However, previous studies revealed similar carbon-carbon interflavanoid linkage types, such as 3,8’-coupled dimer of sakuranetin [Peak uranetin .18 ([M − H]− at m/z 285), 19 ([M − H]− at m/z 301), 21 ([M − H]− at m/z 269) and 22 ([M − H]− at m/z 285) were identified as luteolin , quercetin , apigenin  and kaempferol , respectively. They were further confirmed by comparing the retention time and the MS/MS spectra with the corresponding standards. Peaks 23 and 25 had the same deprotonated molecular ion at m/z 301 and some other characteristic fragment ions, such as the ions at m/z 151 and m/z 125 produced by the RDA cleavage of C ring, the neutral loss of H2O at m/z 283 and the dissociation of B ring at m/z 179. Hence, Peaks 23 and 25 were tentatively identified as the isomers of Peak 19. As for Peak 26, the [M − H]− ion at m/z 299 was obtained, which further generated MS/MS fragment ions [M − H − CH3]− at m/z 284, [M − H − CH3 − CO]− at m/z 256, [M − H − CH3 − CO2]− at m/z 240 and [M − H − CH3 − CO − CHO]− at m/z 227. Compared with those ESI-MS/MS spectra and fragmentation pathways, Peak 26 was thus identified as rhamnocitrin  [32 presented the [M − H]− ion at m/z 359, and its MS/MS fragment ions at m/z 285, 267, 241 and 223 were similar to that of Peak 18, which suggested Peak 32 as a luteolin derivative.For flavones, Peaks m/z 300) ,31. PeakR. davurica. For a better illustration, we classified them into two groups: C-glycosides and O-glycosides, which were identified by comparing their MS/MS spectra with those of corresponding standards and the literature reported previously.According to C-glycosides from R. davurica, the MS/MS spectra and proposed fragmentation pathways are shown in −, [M − H − 90]− and [M − H − 120]− are the characteristic diagnostic ions of glycone [1 and 2 showed the same [M − H]− ion at m/z 447 and MS/MS spectra at m/z 357, 327, 297 and 285, implying that they were structural isomers. Fragment ions at m/z 357 and 327 were due to the collision-induced dissociation (CID) of glycone at the 0−3 bond  and the 0−2 bond . Taken into consideration both the deprotonated molecular ions at m/z 447 and the aglycone ion at m/z 285 , they were tentatively characterized as luteolin monohexoside. For Peak 1  was more abundant than that of [M − H − 120]− at m/z 327 . However, for Peak 2 , was of higher abundance than that of [M − H − 90]− at m/z 357 . Hence, Peaks 1 and 2 were identified as orientin  and isoorientin (luteolin 6-C-glucopyranoside) − at m/z 341, characteristic of C-8-hexosides , and further MS/MS ions at m/z 283 and 269, Peak 3 was thus identified as vitexin  −) and the radical aglycone ions ([Y0 − H]−) −) . At the yl group .4 and 11 exhibited the same [M − H]− ion at m/z 463 and the aglycone ion at m/z 301 , indicating that they were quercetin monosaccharides. Additionally, the aglycone ions of quercetin showed distinct relative abundances of [Y0]− at m/z 301 and [Y0 − H]− at 300. By comparing its LC-MS/MS with the standard compound of isoquercitrin, Peak 4 was identified as isoquercitrin . Peak 11 could thus be deduced as quercetin 7-O-glucoside according to a previous study −). Considering the MS/MS fragments of [Y0]− at m/z 285 and [Y0 − H]− at 284, Peaks 6 and 17 were identified as astragaloside  and kaempferol 7-O-glucoside − at m/z 299 (the loss of a hexose moiety), suggesting that it was a diosmetin monosaccharide. Meanwhile, the presence of the ion at m/z 284 was yielded due to the loss of the methyl moiety, and its glycosylation took place at C-7. Therefore, Peak 5 was tentatively identified as diosmetin 7-O-glucoside [7 also showed the [M − H]− ion at m/z 447 and the aglycone ion at m/z 285 ; while the neutral losses of 120 Da and 146 Da of MS/MS fragments at m/z 327 and 301 indicated that Peak 7 was luteolin 5-O-glucoside [R. davurica for the first time in this work.Peaks 10 exhibited the deprotonated molecular ion at m/z 445 with a [Y0]− at m/z 283 (loss of a hexose moiety). The ion at m/z 268 corresponds to the loss of a methyl moiety (15 Da). By comparing its MS/MS spectra with a reported study, Peak 10 was tentatively identified as physcion 8-O-glucoside [31 ([M − H]− at m/z 283), several fragment ions at m/z 268, 239, 211 and 195 were obtained. Fragment ions of [M − H − 15]− at m/z 268, [M − H − 15 − 29]− at m/z 239 and [M − H − 15 − 29 − 44]− at m/z 195 were produced by the successive loss of the methyl moiety (15 Da), neutral CO + H moiety (29 Da) and CO2 moiety (44 Da). Besides, the fragment ion of at m/z 211 was regarded as the neutral loss of CO moiety (28 Da) from [M − H − CH3 − COH]− at m/z 239. As a result, Peak 31 was determined to be physcion , whose fragment ions were also consistent with those observed using ESI-MS/MS cyclopentapyran, which was similar to catalpol aglycone [R. davurica.Peak rivative . As for aglycone . Moreove2 ≥ 0.9990). In total, 32 components can be classified into five groups based on the structural identifications above. Hence, the individual component contents in R. davurica were quantified by using the corresponding standards for calibration for each group , and their corresponding concentrations were calculated as shown in R. davurica. Due to the chemical diversities of different groups, flavonoid glycosides exhibited higher content of 40.98%, followed by the flavone aglycones of 26.26%, anthraquinones and their O-glycosides of 26.2%, respectively. In general, vitexin was the most abundant glycoside with 86.07 mg/100 g DW (dry weight); aromadendrin and question, the major components of aglycones and anthraquinones, accounted for 21.48 mg/100 g DW and 42.17 mg/100 g DW. In addition, the other components in trace quantities also showed significant variance in the constitutes shown in R. davurica.According to the HPLC screening data summarized in R. davurica has long been used as a kind of folk remedy in many Asian countries. According to the quantification above, flavonoids accounted for the majority of the chemical compositions and represented the dominant class of components in R. davurica. Flavonoids ubiquitously exist and are widely consumed as the important secondary metabolites from natural plants and have remarkable anticancer activities. However, there is very little study on their anticancer activities.The bioactive evaluations in one plant species depend on both the qualitative and quantitative knowledge on this species . As we kR. davurica on human cancer cell lines, like HT-29 and SGC-7901 cells. As shown in R. davurica exhibited significant dose-dependent antiproliferative activities against HT-29 and SGC-7901 cells with IC50 values of 24.96 ± 0.74 and 89.53 ± 4.11 μg/mL, respectively. Meanwhile, R. davurica in a time-dependent manner from 24 h–96 h at a dose of 150 μg/mL, although there was a decrease on SGC-7901 cells at the time from 72 h–96 h. Considering the drug safety, the evaluation of the general toxicity of R. davurica on the normal human hepatic cells (L-O2) was also conducted . Formic acid (FA) and acetonitrile (ACN) of HPLC grade were provided by TEDIA Company Inc. . Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Gibco . Sulforhodamine B (SRB), glutamine, trichloroacetic acid, streptomycin, penicillin and DMSO were purchased from Sigma-Aldrich . Polyamide was obtained from an industrial chemical company affiliated to Nan Kai University . Water for HPLC-UV and ESI-MS/MS was prepared with EPED . All other chemicals and solvents were of analytical grade.R. davurica originated from the northeast of China and were provided by Jikang Pharmaceutical Co., Ltd. . Those raw materials were dried at 40 °C and powdered using a high speed disintegrator, then packed in sealed polyethylene bags and stored in a refrigerator at 4 °C until use. The authentication and identification of the specimens was kindly assisted by the taxonomist (Guangwan Hu) of Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture , Chinese Academy of Sciences. A voucher specimen (No. 0031) was deposited in the herbarium of the Key Laboratory.The barks of R. davurica were accurately weighed and then extracted in an ultrasonic bath  with 60% ethanol for 30 min at room temperature, and the residue was re-extracted twice as described above. Next, the extracts were combined and filtered. After that, the filtrates were concentrated in a rotary evaporator under reduced pressure at 40 °C to afford the crude syrup residues. Later, the crude extracts were dispersed in water (100 mL) and subjected to liquid-liquid partition with petroleum ether  and ethyl acetate (EA), successively. Afterwards, the EA layers were concentrated and fractionated by a polyamide column (45 cm × 5.3 cm). The column was eluted with distilled water to nearly colorless to remove water soluble impurities [First, 100 g raw powered sample of purities  and then2 and 90% relative humidity (RH) under 37 °C. The number of living cells was assessed using a hematocytometer and phase-contrast microscopy. Finally, the cells over 80% confluence (growth phase) were used for the following cell antiproliferation assay [Human cancer cell lines of HT-29 (colon carcinoma) and SGC-7901 (gastric carcinoma) were obtained from the China Center for Type Culture Collection (CCTCC). Cells were routinely grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM) and 1% penicillin (100 U/mL)-streptomycin (100 µg/mL). Furthermore, the cell lines were sub-cultured twice a week, and incubated in a humidified atmosphere with 5% COon assay .4 cells per well. After incubation at 37 °C in 5% CO2 and 90% relative humidity for 24 h to resume exponential growth and stabilization, the culture medium was removed carefully, and an aliquot of 100 μL R. davurica extracts was added into each well in the plates. All of the test samples were first dissolved in DMSO and further diluted with the medium to the final DMSO content less than 0.1%, which was innocuous on cell growth and proliferation. After incubation for another 72 h, cells were fixed with 50 μL 10% cold (4 °C) trichloroacetic acid (TCA) for 30 min at 4 °C. Afterwards, the supernatants were washed out with deionized water five times and air dried at room temperature. Subsequently, the dried plates were stained with 100 μL 4 mg/mL SRB in 1% acetic acid solution for 30 min at room temperature. Next, the plates were washed five times with 1% acetic acid to remove the unbound SRB and then air dried overnight. The protein-bound SRB was solubilized with 150 μL of 10 mM Tris base (pH 10.5), and the plates were left on a gyratory shaker for 10 min. The complete medium with less than 0.1% DMSO was used as the control. The optical density (OD) value of each well was determined with a 96-well plate reader (Tecan) at a wavelength of 540 nm. The inhibition rate (%) was calculated using the equation: inhibition = (ODC − ODT)/ODC × 100%, where ODC and ODT were the OD values of controls and samples of R. davurica, respectively. Each test sample solution was performed in triplicate, and the results were expressed as the means ± SD (standard deviation).The antiproliferative activities against HT-29 and SGC-7901 cells were estimated by means of the protein-staining sulforhodamine B (SRB) microculture colorimetric assay with some modification . The assR. davurica were carried out using a Thermo Accela 600 HPLC system . The chromatographic separation was performed on a Waters SunFire™ RP-C18 column . The mobile phases were composed of 0.1% aqueous formic acid (A) and acetonitrile (B). An aliquot of a 10-μL sample solution was injected into the HPLC system, and the linear eluting gradient was as follows: 20% B in 0–2 min, 20%–45% B in 2–15 min, 45%–70% B in 15–31 min and 70% B in 31–35 min. The column temperature was maintained at 30 °C. The flow rate was 0.4 mL/min, and the online UV spectrum was monitored at the wavelength of 360 nm.Experiments for phytochemical fingerprint analysis of n mode to obtain fragmentation. The negative ionization mode was applied, and the optimized instrument settings were set as follows: source voltage, 3.0 kV; cone voltage, 40.0 V; desolvation temperature, 350 °C; capillary temperature, 250 °C; nebulizing gas flow rate, 6.0 L/min; sheath gas (N2) pressure, 40 arb; Aux gas (N2) pressure, 10 arb; collision energy (CE), 10 V; collision energy grad (CE grad), 0.035 V/m. Mass spectra data were obtained with the full-scan mode for m/z in the range from 150 to 1500, and the nine most abundant ions were selected for the further MS2 spectra. All data acquisition and analysis were performed using the Thermo Xcalibur ChemStation (Thermo Fisher Scientific).The ESI-MS/MS analysis was carried out using a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific) equipped with an ESI source operating in Auto-MSR. davurica by HPLC. Each standard was accurately weighed, prepared in methanol, and then, the calibration curves were established by diluting the standard stock solutions into a series of concentrations measured at 360 nm. Six different concentrations (1.0–333 μg/L) of each standard were used for the calibrations and measured in triplicate. Method validations included accuracy, correlation determination (R2), limit of detection (LOD), limit of quantitation (LOQ), linearity range, repeatability, precision and recovery. Finally, the quantification of each individual compound was calculated based on the calibration curves of the standards for the corresponding HPLC peak area values of R. davurica. In the present study, individual components are presented as mg per 100 g DW, and the total content was defined as the sum of each corresponding quantified component.For the quantitative analysis, three representative standards  were applied to calculate the individual component concentration present in R. davurica. As a result, 32 peaks were detected. Structural identifications and characterizations showed that 23 of them were identified as flavonoids based on the comparisons of their ESI-MS/MS data with the corresponding standards and fragmentation pathways reported in the previous literature. Among 32 components identified, including orientin (Peak 1), isoorientin (Peak 2), diosmetin 7-O-glucoside (Peak 5), astragaloside (Peak 6) and luteolin 5-O-glucoside (Peak 7), 14 components were identified in R. davurica for the first time. For individual component quantitative analysis, flavonoid glycosides, flavone aglycones and anthraquinones exhibited higher content of 40.98%, 26.26% and 26.2%, respectively, which consisted of the major components of R. davurica. For the discrepant contents of individual components, vitexin, aromadendrin and question accounted for 86.07, 24.18 and 42.17 mg/100 g DW, respectively. Meanwhile, to better correlate the phytochemical components with their pharmacological activities, the antiproliferative activities against human cancer cells were tested in vitro and exhibited distinct inhibitory effects against HT-29 and SGC-7901 cells with IC50 values at 24.96 ± 0.74 and 89.53 ± 4.11 μg/mL, respectively. Finally, R. davurica displayed a much higher IC50 at 229.19 ± 8.52 μg/mL on L-O2, which suggested that R. davurica showed very low or no toxicity on hepatic cell viability. The current study revealed for the first time the correlations between the flavonoids of R. davurica with their antiproliferative activities, which indicated that the fingerprint profile of flavonoids and their anticancer activities could provide valuable information on the quality control for this herbal medicine and its derived natural remedies.In this study, a HPLC-ESI-MS/MS method was employed to comprehensively analyze the phytochemical fingerprint profile of


G. madruno, one of which, 7''-O-(6''''-acetyl)-glucoside of morelloflavone, is a new compound identified on the basis of 1D, 2D NMR (HMQC and HMBC) spectroscopic methods and chemical evidence. The antioxidant activity of the biflavonoids against low-density lipoprotein (LDL) peroxidation induced with Cu2+, was studied by means of a TBARS assay. The antioxidant potential of a biflavonoid fraction (BF) was also evaluated and correlated with its biflavonoid content. The flavanone-(3→8'')-flavone biflavonoids displayed antioxidant activity, particularly morelloflavone, which was significantly more potent than quercetin, with a CE50 of 12.36 μg/mL. Lipid peroxidation, was also significantly reduced in the presence of the BF (EC50 = 11.85 μg/mL). These results suggest that the BF is an excellent antioxidant.Six biflavonoids were isolated from  Garcinia madruno (Kunth) Hammel, commonly known as madroño, is a tree endemic to Central and South America. It is resistant to plagues and illnesses and adaptive to different environmental conditions 288 +67,394, [θ]351 +36,263 (MeOH); UV λ (nm): 332, 292, 209, MeOHmax; IR (KBr) νmax cm−1: 3,168, 1,648, 1,536, 1,316; 1H-NMR and 13C-NMR see m/z 761.1720 [M+H]+ .Brown powder; m.p. 268–270 °C; The protective effect of the biflavonoids and the BF against LDL-peroxidation was determined by a TBARS assay.® .50 mL of blood was collected by venepuncture into heparinized tubes from healthy non-smoking volunteers (20–25 years old). Plasma was recovered by differential density ultracentrifugation at 2,500 rpm and 4 °C, in a Beckman XL-100 ultracentrifuge  equipped with a SW-55Ti rotor, as described elsewhere. The LDL fraction was obtained by centrifugation with 1.6 mL of NaCl (17 M) in distilled water at 49,500 rpm for 12 h. The superior fraction was removed and 1.6 mL of KBr (10 M) was added before another centrifugation for a period of 18 h. SDS-PAGE was used to confirm the purity of the collected fractions . The concentration of protein was determined by the Protein Quantification Kit-Rapid method of Fluka4 100 μM, and finished by 5 μL of 1% EDTA and cooling. A TCA–TBA–HCl stock solution  was added to the reaction mixture. The solution was then heated at 95 °C for 60 min. The supernatant was filtered through a 0.45 μm membrane and a reading was made at 532 nm. Readings of three independent experiments were carried out. One-way ANOVA with a Student Newman-Keuls post-test was performed using GraphPad Prism version 4.00 and p < 0.05 was considered as significant difference.The formation of products from peroxidation of LDL was determined by the thiobarbituric acid reactive substances assay (TBARS). The LDL was incubated at 37 °C in 0.1 M potassium phosphate buffer, and made up to a final protein concentration of 300 μg/mL. Volumes of 50 μL of biflavonoids or quercetin (positive control) at different concentration were added and the peroxidation was initiated by 50 μL of CuSOG. madruno. Compound 5 was found to be a new biflavonoid glycoside on the basis of spectroscopic analyses and chemical evidence. Some of the compounds exhibited potent lipid peroxidation inhibition activity, and lipid peroxidation was also significantly reduced in the presence of the biflavonoid fraction (BF), mainly due to morelloflavone. These results suggest that the BF is an excellent antioxidant.In summary, six biflavonoids were isolated from


Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.The  Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.The thermophilic biomass-degrader IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications. They aron xylan , 5, 6, bon xylan , 8. The lization , 10–12.LPMOs play a vital role in the decomposition of plant biomass in nature and have gained importance in industrial processes aimed at the degradation of recalcitrant polysaccharides to fermentable sugars –15. In t–20–β-1,4-linked plant cell wall polysaccharides, including (insoluble) cellulose as well as (soluble) xyloglucan, glucomannan, and cellooligosaccharides  FCH 10.5, namely, mclpmo9b, mclpmo9e, mclpmo9f, and mclpmo9h, were upregulated during growth on wheat bran compared to that on glucose. Furthermore, mclpmo9f and mclpmo9h were also highly upregulated on beechwood xylan as the sole carbon source , suggesting involvement in xylan degradation. Since M. cinnamomea FCH 10.5 showed poor growth on cellulosic substrates but substantial growth on hemicelluloses like xylan and glucomannan   indicated that only McAA9A was N-glycosylated, which agrees with it having predicted N-glycosylation sites at Asn160 and Asn208. Binding assays with Avicel showed similar weak binding for all five proteins, in accordance with the absence of carbohydrate-binding modules (CBMs) (File S1b). Four of the purified enzymes, McAA9A, -B, -F, and -H, produced H2O2 in the presence of ascorbic acid but not when the reducing agent was omitted . This oxidase activity is a futile side reaction of LPMOs  , we hypoof LPMOs , and theMcAA9s were found to be quite diverse, with sequence identities of the catalytic AA9 domains ranging from 29% to 61% (File S1c). Phylogenetic analysis of the catalytic domains showed that the McAA9s do not cluster together but are spread out among other characterized AA9 LPMOs . While about 20% of known AA9 LPMOs contain a CBM  that might form a second disulfide bond, which is only present in about one-third of the enzymes in the multiple-sequence alignment (MSA) . Homology models generated with Phyre2 -like β-sandwich fold and a histidine brace motif that holds the copper ion in place  in the presence of ascorbic acid as an electron donor. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis showed that McAA9A, -B, and -F produced native as well as C1-, C4-, and C1/C4-oxidized cellooligosaccharides from PASC  further confirmed the presence of native and oxidized products . Both HPAEC chromatograms, with large peaks for the C4-oxidized products, and the mass spectra, showing small signals for the diagnostic sodium salts formed by C1 oxidized products, indicate that these three LPMOs predominantly oxidize cellulose at the C4 position.Since all previously characterized AA9 LPMOs are active on cellulose, the four   and hydrated oxidized (Δm/z +16) xylan fragments, indicating the formation of aldonic acid or gemdiol species, resulting from C1 or C4 oxidation. The presence of sodium salts of the aldonic acids of non-GlcAOMe-containing xylooligosaccharides (Δm/z +38) confirmed the occurrence of C1 oxidation, but the data did not allow conclusions about the ratio between C1 and C4 oxidation of xylan.In contrast to the other three tested rom PASC , 34. ConMcAA9s were incubated with tamarind xyloglucan  and birchwood xylan . In standard assays, McAA9A, -B, and -F, but not McAA9H, were active toward TXG . Most interestingly, however, coating of cellulose fibers with TXG or BX changed the susceptibility of the hemicelluloses to degradation by the McAA9s. McAA9H, which was completely inactive on TXG alone, generated products from TXG in reaction mixtures containing both cellulose and TXG  could be detected, indicating that McAA9H is able to oxidize the C1 position upon cleavage of xyloglucan.No activity was detected for any of the  and TXG , in addiMcAA9s were, to differing degrees, active toward BX-coated cellulose and produced cellooligosaccharides (from PASC) as well as native and oxidized xylooligosaccharides (from BX or PASC). For McAA9H, signals corresponding to oxidized xylooligosaccharides (with or without GlcAOMe) were dominating, corroborating the observations for the reactions with PASC alone, which strongly suggested that this enzyme strongly prefers xylan as the substrate .Substrate conformation also seemed to influence activity toward xylan, since all four ubstrate . On the McAA9A and McAA9F, showed clear activity on cellooligosaccharides (DP6), generating C4 oxidation products .Two of the tested LPMOs, McAA9A, displayed trace activity on lichenan, a linear β-1,3-β-1,4-glucan with a 1:2 ratio of β-1,3 to β-1,4 linkages . None of the McAA9s was active on ivory nut mannan (INM), a linear β-1,4-linked mannan without substitutions, while McAA9A was able to depolymerize konjac glucomannan (KGM), a linear β-1,4-linked mannan with randomly distributed glucosyl residues (∼35%)  . The MS M. cinnamomea is a thermophilic, hemicellulose-degrading fungus that grows abundantly on xylan-rich substrates. In our study, we characterized four of the eight AA9s encoded by the fungus  and found that the substrate preference of the organism    is refleaterial) . McAA9H ubstrate .Lentinus similis (LsAA9A) displayed activity toward xylooligosaccharides, although it was at two magnitudes lower than activity on cellooligosaccharides  acted on both cellulose and hemicelluloses equally well, but the available data do not allow an assessment of substrate preferences and background activities in the absence of ascorbic acid were very high in this study . TherefMcAA9s studied so far, considering that, previously, only a few LPMOs (two AA9s and two AA14s) have been reported to be able to cleave xylan. Of note, for two of the five M. cinnamomea enzymes, McAA9A and McAA9B, the xylan activity was weak and could easily have been overlooked. Most studies so far indicate that LPMO activity on xylan is strongly linked to the presence of cellulose, indicating the importance of xylan conformation for susceptibility to LPMOs. Xylan exists in a 3-fold helical screw conformation in solution but flattens into a 2-fold screw ribbon when bound to cellulose microfibrils in the plant cell wall  and upon binding to cellulose microfibrils   shows considerable differences between the surfaces around the active site. The functional implications of these differences would be worth further studies.While a flat conformation is suggested to be necessary for sufficient interaction with the LPMO active site, several AA9 LPMOs, including rmation) . Unlike ellulose  and d. TM. cinnamomea presents a good model to investigate the specificities of an organism’s full complement of LPMO enzymes. We show here that the differential transcriptional regulation of AA9s previously studied in M. cinnamomea during growth on glucose, wheat bran, and beechwood xylan . The recently characterized McAA9G   .McAA9s, the slow growth of M. cinnamomea on cellulosic substrates points to a group of LPMOs that are not used primarily for breaking down cellulose. In their study of xylan-active AA14 LPMOs, Couturier et al. noted that AA14s are primarily present in white- and brown-rot fungi that specifically target the protective heteroxylan-rich layer covering cellulose microfibrils in wood  (Escherichia coli strain Stellar (TaKaRa) and Pichia pastoris strain SMD1168H (Invitrogen) were used as hosts for subcloning and heterologous expression of recombinant proteins, respectively. 143691)  was usedM. cinnamomea was grown in liquid basal medium containing 4 g liter−1 KH2PO4, 13.6 g liter−1 (NH4)2SO4, 0.8 g liter−1 CaCl2·2H2O, 0.6 g liter−1 MgSO4·7H2O, 6 g liter−1 Bacto peptone, 10 mg liter−1 FeSO4·7H2O, 3.2 mg liter−1 MnSO4·H2O, 2.8 mg liter−1 ZnSO4·7H2O, 4 mg liter−1 CoCl2·6H2O, and 200 ml liter−1 Tween 80. The pH was adjusted to 5.8, and 1% (wt/vol) of a carbon source  was added before autoclaving. Volumes of 125 ml liquid medium in baffled Erlenmeyer flasks (500 ml) were inoculated with a spore suspension prepared from fresh malt extract agar (MAE) plates. Cultures were grown at 45°C for 19 days with shaking at 250 rpm, and samples were taken at regular intervals.http://www.ibi.vu.nl/programs/pralinewww/), Clustal Omega , and MEGA7 software were used for multiple-sequence alignment (MSA) of AA9 proteins. Phylogeny.fr (http://www.phylogeny.fr/) and iTOL (https://itol.embl.de/) were used for generating phylogenetic trees. A detailed list of proteins used in the alignments is provided in File S1d. For homology modeling, the online tool Phyre2 was used with default parameters  and were named according to current naming conventions: mclpmo9A (locus tag MALCI_0000123.8), mclpmo9B (MALCI_0000604.52), mclpmo9C (MALCI_0000604.99), mclpmo9d (MALCI_630.43), mclpmo9e (MALCI_0000639.3), mclpmo9f (MALCI_0000739.16), mclpmo9g (MALCI_0000780.38), and mclpmo9h (MALCI_0000638.10) (File S1f and g). Total RNA was extracted from M. cinnamomea FCH 10.5 grown in fungal basal medium containing 1% wheat bran as the sole carbon source for 48 h, as described previously  before being used in the In-Fusion cloning reaction  with PCR amplicons according to the manufacturer’s protocol. Supplied competent E. coli Stellar cells were transformed by heat shock with 2 μl of In-Fusion mix and positive transformants selected on medium containing zeocin. The presence of recombinant genes was checked by colony PCR, and completeness was verified by sequencing (Eurofins).Target genes were amplified from cDNA in 20-μl PCRs using the Phusion high-fidelity PCR kit  with the following components: 4 μl 5× HF buffer, 0.4 μl 10 mM dNTPs, 0.5 μM each primer, 0.8 μl cDNA synthesis product, 0.2 μl Phusion polymerase. PCR cycle conditions were 98°C for 2 min; 30 cycles of 98°C for 10 s, 60°C for 30 s (−0.5°C every cycle), and 72°C for 1 min; and 72°C for 10 min for the final extension. Amplification of the expected products was verified on agarose gel electrophoresis, and DNA fragments were extracted by a GeneJET gel extraction kit . Primers were designed to contain 3′ and 5′ flanking regions homologous to the desired insertion points in the pPICZαA vector, as well as BstBI and SalI restriction enzyme recognition sites. Since the N-terminal histidine is essential for LPMO functionality, P. pastoris SMD1168H (Invitrogen) was performed, as described in the manufacturer’s manual, with MssI-linearized pPICZαA recombinant plasmids. Zeocin-resistant transformants were used for small-scale protein expression in 96-well deep-well microplates. The presence of recombinant protein was confirmed by SDS-PAGE, and the clones with the highest expression levels were used for large-scale protein production in a fed-batch process in bioreactors (Infors) according to the Pichia fermentation process guidelines (Invitrogen). Briefly, 500 ml fermentation basal salts medium containing PTM1 trace salts, 4% glycerol, and 0.003% antifoam was inoculated with 50 ml P. pastoris preculture. The pH was kept at 5.6 with 28% ammonium hydroxide throughout the whole fermentation. After about 24 h of batch growth, glycerol feed was initiated for 24 h until wet cell weight was above 180 mg ml−1. Gene expression under the control of the AOX1 promoter was induced by switching to a 100% methanol feed, first at a low feed rate (1 to 3 ml h−1) for several hours to allow for cell adaptation and then a higher rate (2 to 6 ml h−1). Dissolved oxygen values were kept above 20% by controlling the feed rate, agitation, and aeration. After 4 to 5 days of induction, culture broth containing the secreted recombinant protein was separated from the cells by centrifugation and filtering.Transformation of competent McAA9A, -B, and -F) or in combination with hydrophobic interaction chromatography (HIC) (McAA9C and -H) on an ÄKTA system . For AEX, culture broth was concentrated and buffer exchanged to 20 mM Tris-HCl, pH 8.0, using a cross-flow filtration capsule  with a 10-kDa-cutoff polyethersulfone (PES) membrane. To purify enzymes using AEX, a HiTrap QLX 1-ml column  was used with start buffer  and elution buffer  at a flow rate of 1 ml min−1 and a gradient of 0 to 100% B over 60 min. Fractions containing target protein were subjected to a further step of HIC with a HiLoad 16/10 phenyl Sepharose column  using start buffer  and elution buffer  with a flow rate of 2 ml min−1 and a stepwise gradient of 40%, 80%, and 100% B. Proteins with electrophoretic purity were concentrated using 3-kDa-cutoff spin columns , buffer exchanged to 20 mM sodium citrate buffer, pH 6.0, and stored in aliquots at –20°C until further use. The concentration of purified proteins was determined spectrophotometrically by NanoDrop using calculated molecular mass and molar extinction coefficients derived from the sequence. The purified enzymes were treated with EndoHf (New England Biolabs) under denaturing conditions, according to the manufacturer’s instructions, and analyzed using SDS-PAGE to determine N-glycosylation.Protein purification was done by anion-exchange chromatography (AEX) alone (Neurospora crassa LPMO9C (NcLPMO9C) (Thermoascus aurantiacus LPMO9A (TaLPMO9A)  at defined time points, and the reaction was stopped by adding 50 μl 0.2 M HCl. Before analysis, all samples were filtered with 0.45-μm-pore-size 96-well filter plates (Millipore) and a vacuum manifold.The following substrates were used: phosphoric acid swollen cellulose   and TheraLPMO9A) , both pr−1 HRP, 0.05 mM ascorbic acid (AscA), and 0.4 to 3.3 μM enzyme in 50 mM sodium citrate buffer, pH 6.0. The reaction was measured at 30°C for 30 min on a FLUOstar Omega microplate reader by recording fluorescence  every 60 s. A standard curve was prepared with H2O2 (0.25 to 5 μM).Generation of hydrogen peroxide by LPMOs was detected using the Amplex Red assay , which e−1 with the following gradients: 0 to 10 min, 0 to 10% B (curve 5); 10 to 35 min, 10 to 30% B (curve 5); 35 to 40 min, 30 to 100% B (curve 6); 40 to 41 min, 100 to 0% B (curve 6); 41 to 50 min, 100% A (curve 5). For analysis of hemicellulosic substrates, a 75-min gradient  and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Analysis with HPAEC-PAD was performed on a Dionex ICS5000 instrument equipped with a CarboPac PA1 guard column  and a CarboPac PA1 column . Two mobile phases were used, A (0.1 M NaOH) and B (1 M sodium acetate in 0.1 M NaOH). For analysis of cellulosic substrates, a 50-min gradient  was usedgradient  was used−1 2,5-dihydroxybenzoic acid in 30% acetonitrile and 0.1% trifluoroacetic acid), applied to an MTP 384 ground steel target plate , and air dried. Data were collected with flexControl software (Bruker) and analyzed using mMass software.Analysis by MALDI-TOF was performed on an Ultraflex instrument  equipped with a nitrogen 337-nm laser beam in positive reflector mode, as described previously . A 1.5-μMcAA9s have been deposited in the GenBank database under the following accession numbers: McAA9A, mclpmo9a, MK135883; McAA9B, mclpmo9b, MK135884; McAA9C, mclpmo9c, MK095629; McAA9D, mclpmo9d, MK135885; McAA9E, mclpmo9e, MK135886; McAA9F, mclpmo9f, MK135887; McAA9G, mclpmo9g, MK135888; and McAA9H, mclpmo9h, MK135889.The nucleotide sequences of the genes (genomic DNA) encoding (putative)


Importantly, we did not observe elevation of the ER stress markers p-eIF2α, ATF4, CHOP, p-IRE1α, or BiP at any age in 5XFAD or 5XFAD;BACE1−/− compared with nontransgenic mice. Despite lacking Aβ generation, 5XFAD;BACE1−/− mice still expressed APP and PS1 transgenes, indicating that their overexpression does not cause ER stress. These results reveal the absence of ER stress in 5XFAD mice, suggesting that artifactual phenotypes associated with overexpression-induced ER stress are not a concern in this model.Alzheimer's disease mouse models that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1) form β-amyloid (Aβ) plaques, a hallmark Alzheimer's disease lesion. It has been assumed that the neuroinflammation, synaptic dysfunction, neurodegeneration, and cognitive impairment observed in these mice are caused by cerebral Aβ accumulation. However, it is also possible that accumulation of the overexpressed transmembrane proteins APP and PS1 in the endoplasmic reticulum (ER) triggers chronic ER stress and activation of the unfolded protein response (UPR). The 5XFAD mouse, a widely used amyloid pathology model, overexpresses APP and PS1, displays aggressive amyloid pathology, and has been reported to exhibit ER stress. To systematically evaluate whether 5XFAD mice have increased ER stress, here we used biochemical approaches to assess a comprehensive panel of UPR markers. We report that APP and PS1 levels are 1.8- and 1.5-fold, respectively, of those in 5XFAD compared with nontransgenic brains, indicating that transgenes are not massively overexpressed in 5XFAD mice. Using immunoblotting, we quantified UPR protein levels in nontransgenic, 5XFAD, and 5XFAD;BACE1 Alzheimer's disease (AD)The 5XFAD mouse is one of the most widely used models of amyloid pathology that overexpress transgenes , FloridaBecause of the early onset of pathology and memory impairment, the 5XFAD model is extensively used in AD research; there are currently 391 results when the term “5XFAD” is searched in PubMed. However, concerns have been raised that endoplasmic reticulum (ER) stress caused by transgenic overexpression of the transmembrane proteins APP and PS1 may lead to ER accumulation of mis-folded proteins and activation of the unfolded protein response (UPR), which may be responsible for artifactual phenotypes in the 5XFAD model .The ER is the site of folding for all membrane and secreted proteins. It contains high levels of the chaperone protein BiP/GRP78, which has both a chaperone and a stress-sensing function. Normally, BiP/GRP78 helps fold new proteins in the ER and interacts with the ER stress sensors PERK, IRE1, and ATF6, preventing their activation. When the level of unfolded proteins becomes too high, BiP releases these sensors, which then dimerize  or translocate to the Golgi (ATF6). PERK and IRE1 autophosphorylate, resulting in up-regulation of the transcription factors ATF4, CHOP, and XBP1s, which induce the expression of genes involved in ER-associated degradation, antioxidant responses, amino acid metabolism, and apoptosis. ATF6 is cleaved, releasing ATF6f, which translocates to the nucleus and activates increased transcription of chaperones to help manage the unfolded protein burden . To detect APP, we used an antibody directed against amino acids 750–770 of human APP . As we reported previously . In general, these represent relatively low levels of transgene overexpression compared with other amyloid pathology mouse models such as PDAPP, Tg2576, TgCRND8, and APP23, in which transgenic APP is overexpressed 10-fold  or thapsigargin (B). By observing an increase in ER stress proteins in the lysates of drug-treated versus vehicle-treated neurons, we were able to verify that our antibodies recognized the correct ER stress proteins. Additionally, by comparing the size of the increased protein band in treated neurons with that in brain homogenates, we were able to validate that we were quantifying the correct proteins in vivo, as some proteins such as ATF4 and p-IRE1α are in low abundance in brain homogenates, and some antibodies are not entirely specific.To begin our evaluation of ER stress in 5XFAD mice, we analyzed 4-month-old mice, which have a moderate amount of amyloid pathology and have been expressing 5XFAD transgenes for about 2.5 months. Before starting the study, we validated a panel of antibodies, shown in icamycin A or thapsigargin B. By obsA and B) and observed no significant differences between the two genotypes for the ER stress proteins examined (C). For quantification, the phosphorylated proteins p-eIF2α and p-IRE1α were normalized to their respective total proteins, but all other proteins were normalized to β-tubulin. APP has been shown to confirm genotype and expression of the 5XFAD transgene. There was some animal-to-animal variation, as expected, especially for phospho-proteins, but no statistically significant differences in the means by two-tailed Student's t test were observed.Using the previously mentioned cohort of 4-month-old mice, we performed immunoblot analysis to determine the relative levels of ER stress proteins for the assessment of UPR activation in 5XFAD mice compared with nontransgenic littermates . 5XFAD;BACE1−/− mice, which overexpress APP and PS1 transgenes but do not generate Aβ, allowed us to determine if changes in ER stress occurred, and if so, whether they were caused by amyloid pathology or by prolonged overexpression of APP and PS1. Importantly, we observed no changes in the levels of the ER stress proteins examined at either 6 or 9 months of age in 5XFAD compared with nontransgenic mice . To verify the BACE1 genotype of the mice, we performed BACE1 immunoblots and observed that the enzyme is elevated ∼2-fold at both ages, as reported previously . This method has been validated as an effective and inexpensive approach to determine relative Aβ levels in transgenic mice and human brains . The α- and β-CTFs are barely detectable under these conditions in nontransgenic mice, preventing relative quantification, but the APP β-CTF is the most prominent band in 5XFAD brain homogenates, whereas it is absent in 5XFAD;BACE1−/− mice. Our previous work has shown that, in 5XFAD mice, the β-CTF, like APP transgene expression and Aβ42, was elevated in females compared with males  approval.5XFAD mice were generated as described previously (−/−  mice to l-lysine–coated 10-cm dishes (7.5 × 106 cells/dish) in Neurobasal medium supplemented with 2% B-27, 500 μm glutamine, 10% horse serum, and 2.5 μm glutamate . After 2 h, the medium was changed to Neurobasal with 2% B-27, 500 μm glutamine, and 2.5 μm glutamate. After 1–3 days, the medium was replaced with Neurobasal plus 2% B-27 and 500 μm glutamine. Following 7 days in culture, neurons were treated with 1 μm thapsigargin or DMSO vehicle for 24 h or 5 μg/ml tunicamycin or DMSO vehicle for 5 h. To collect lysates, dishes were rinsed once in PBS, lysed in 300–500 μl of radioimmune precipitation assay buffer, and spun for 10 min at 10,600 × g at 4 °C. The protein concentration of the supernatant was quantified using the BCA assay (Pierce), and the supernatant was used for immunoblotting.Embryonic day 15.5–16.5 C57BL/6 mouse cortical neurons were plated on poly-Snap-frozen hemibrains were homogenized in 1 ml of 1× PBS with 1% Triton X-100 supplemented with protease inhibitors  and Halt phosphatase inhibitor mixture (Thermo Scientific). Protein concentration was quantified using a BCA assay (Pierce). Thirty or fifty micrograms  of brain homogenate and 10 or 20 μg of neuronal lysates were separated on 4–12% BisTris gels (Invitrogen NuPage), and protein was transferred to a 0.45-μm PVDF membrane. For detection of PS1 NT, samples were mixed with standard Laemmli buffer but not boiled prior to electrophoresis . Membranm guanidine hydrochloride (GuHCl) and 82 mm Tris-HCl (pH 8.0)  over three nights on a nutator. Then 1 μl of GuHCl-extracted sample  was spotted in triplicate on a gridded nitrocellulose membrane, dried for 1 h at 37 °C, rinsed in TBS-T  and then water, and stained with Ponceau S. Two identical blots were made and incubated in either 1:4000 anti-Aβ42 rabbit mAb  or 5% milk only (primary delete), followed by horseradish peroxide–conjugated secondary antibody . The duplicate blots incubated with anti-Aβ42 antibody or primary delete were developed together with ECL SuperSignal WestPico (Pierce), imaged simultaneously using a FluorChemR imager (ProteinSimple), and then quantified with Alphaview software (ProteinSimple). The signal from the primary delete blot was subtracted from the Aβ42 signal; this was normalized to Ponceau S staining and the triplicates were averaged, and statistics were performed as described below.For Aβ42 dot blots, 10 mg/ml brain homogenates were extracted in 1.56 volumes of 8.2 t test and ANOVA were done using InStat software  to compare means of the various genotypes and genders. For the scatter plots, * and # indicate groups statistically compared by Student's t test or ANOVA; * 0.05 > p > 0.01 ** 0.01 > p > 0.001 *** 0.001 > p > 0.0001. Error bars indicate standard error. * indicates comparison with the non-Tg group, whereas # indicates comparison with the 5XFAD group.Student's two-tailed K. R. S. and R. V. conceptualization; K. R. S. formal analysis; K. R. S. and J. P. investigation; K. R. S. and R. V. methodology; K. R. S. writing-original draft; K. R. S., J. P., and R. V. project administration; K. R. S. and R. V. writing-review and editing; R. V. resources; R. V. funding acquisition.