TCF3‐HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t‐ALL case, while TCF3‐PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t‐ALL cases. Using 4 t‐ALL and 16 t‐ALL cell lines, drug response profiling was analyzed. t‐ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t‐ALL cell lines. Sensitivities to three , four  and five  agents, widely used in induction therapy, were significantly poorer for t‐ALL cell lines than for t‐ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P‐glycoprotein (P‐gp) were higher in t‐ALL cell lines than in t‐ALL cell lines. Inhibitors for P‐gp sensitized P‐gp‐positive t‐ALL cell lines to VCR and DNR. Knockout of P‐gp by CRISPRCas9 overcame resistance to VCR and DNR in the P‐gp‐positive t‐ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P‐gp‐positive t‐ALL cell line. These findings indicate involvement of P‐gp in resistance to VCR and DNR in Pgp positive t‐ALL cell lines. In all four t‐ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t‐ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t‐ALL.t, responsible for TCF3‐HLF fusion, is a rare translocation in childhood B‐cell precursor acute lymphoblastic leukemia(BCP‐ALL). t, producing TCF3‐PBX1 fusion, is a common translocation in childhood BCP‐ALL. Prognosis of t‐ALL is extremely poor, while that of t‐ALL has recently improved dramatically in intensified chemotherapy. In this study,  Prior to analyses of the cell lines, we prospectively examined the levels of minimal residual disease (MRD) during induction therapy in a newly diagnosed t‐ALL case and t‐ALL cases. We confirmed high level of MRD in the t‐ALL case, indicating that ‐ALL shows resistance to chemotherapeutic agents in induction therapy. To verify this idea, we analyzed in vitro sensitivities to multiple chemotherapeutic agents of the t‐ALL and t‐ALL cell lines in induction therapy. We established that t‐ALL cell lines were significantly more resistant to VCR, daunorubicin (DNR), and glucocorticoids than t‐ALL cell lines. Furthermore, we confirmed comprehensive drug resistance to multiple agents in t‐ALL cell lines in induction therapy.22.1TCF3‐PBX1 was performed using sense (5′‐CCAGCCTCATGCACAACCA‐3) and antisense (5’‐ GGGCTCCTCGGATACTCAAAA‐3′) primers with probe (5′‐FAM‐CCCTCCCTGACCTGTCTCGGCC‐TAMRA‐3′), as previously described.2, 0.01 U Uracil DNA glycosylase per µL, 0.025 U of AmpliTaq Gold per microliter, and 1 × TaqMan PCR buffer . Amplification and detection were performed using an ABI 7900 sequence detection system . The data were analyzed by Sequence Detector version 1.63 software (Applied Biosystems). The final results were normalized by the amount of internal control GAPDH (Thermo Fisher Scientific). Real‐time PCR analysis for TCF3/HLF was performed using sense (5′‐GCCTCATGCACAACCACGCG‐3) and antisense (5′‐CCCGGATGGCGATCTGGTTC‐3′) primers with a SYBR Green PCR Master Mix (Applied Biosystems). As an internal control for TCF3/HLF quantification, ACTB was quantified using sense (5'‐ACCTTCTACAATGAGCTGCGT‐3') and antisense (5'‐GTACATGGCTGGGGTGTTGA‐3') primers.The patients were enrolled in Tokyo Children's Cancer Study Group (TCCSG) L04‐16 study.2.2MLL‐rearranged (MLL+) ALL cell lines , six Philadelphia chromosome (Ph)‐positive ALL cell lines , and eight other ALL cell lines  were used ‐ALL cell lines  and 16 t‐ALL cell lines  were used in this study.‐ALL celled Table .20 All c2.350s of DNR, VCR, prednisolone (Pred), dexamethasone (Dex), l‐asparaginase (l‐Asp), cyclophosphamide (CPM), and selumetinib, an alamarBlue assay was performed.5) were plated onto a 96‐well flat‐bottom plate in triplicate in the absence or presence of seven concentrations of each drug. The cells were cultured for 44 hours to determine the DNR, VCR, and CPM sensitivities and for 68 hours to determine Pred, Dex, l‐Asp, and selumetinib sensitivities, and, then, 20 µL of alamarBlue was added. After a 6‐hours additional incubation with alamarBlue, absorbance at 570 nm was monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of the treated wells to that of the untreated wells as a percentage. The concentration of agent required to reduce the viability of the treated cells to 50% of the untreated cells was calculated, and the median of three independent assays was determined as IC50. The median of the IC50s measured by three independent assays was determined.To determine IC2.4To detect apoptotic events, cells were cultured in the absence or presence of DNR or VCR in combination with or without verapamil, cyclosporine A (CyA), or nilotinib for 24 hours, and stained with a fluorescein isothiocyanate‐conjugated Annexin‐V  and actinomycin‐D . Cell surface expression of P‐gp was analyzed using a phycoerythrin‐conjugated anti‐P‐gp antibody. For the functional assay of P‐gp‐mediated efflux of calcein‐AM (CAM), HALO1 cells were incubated with 0.25 mmol/L of CAM for 10 minutes at 37°C in the absence or presence of velapamil, CyA, or nilotinib. The stained cells were analyzed by flow cytometry .2.5l‐Asp), four , and five  drugs were analyzed according to previous reports.50 values; seven cell lines), intermediately sensitive , or resistant  to each drug. A sensitive result was rated as 1, an intermediate result as 2, and a resistant result as 3, and the total score was calculated by adding these counts.The combined sensitivities of 4 t‐ALL and 16 t‐ALL cell lines to three , reverse transcription was performed using a random hexamer  by Superscript II reverse transcriptase (Invitrogen), and then incubation with RNase (Invitrogen). For quantitative real‐time PCR, triplicated samples containing cDNA with TaqMan Universal PCR Master Mix (Applied Biosystems) and Gene Expression Product listed in Table 2.74) were injected into the tail vein to establish xenografts. One day after injection of HALO1 cells, each of the five mice were treated with 0.5 mg/kg of DNR alone or 0.5 mg/kg of DNR in combination with 50 mg/kg of CyA for five consecutive days. DNR and CyA were further diluted with phosphate‐ buffered saline (PBS) and intraperitoneally injected into the mice. CyA was injected one hour before administration of DNR. The control group of five mice was administered PBS only.Six‐week‐old female NSG (NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ) mice were purchased from Jackson Laboratory . The experiment was performed in a specific pathogen‐free unit after approval of protocols for animal care and experiment by the Tokyo Medical and Dental University animal care and use committee (approved No. A2017113). HALO1 cells (1 x 102.8http://crispr.mit.edu), and the synthesized oligomers were cloned into CRISPR/Cas9 vectors . Three days after electroporation of the ABCB1‐targeting CRISPR/Cas9 vector into HALO1 cells using the Neon electroporation transfection system (Thermo Fisher Scientific), CD4‐positive cells were selected using CD4 microbeads  and expanded for further analyses.To knockout P‐glycoprotein  expression with the CRISPR/Cas9 system, we selected 5′‐TTTGGCTGCCATCATCCATGG‐3′, which showed the highest off‐target hit score in the CRISPR design tool . Libraries were prepared using the HaloPlex Target Enrichment System (Agilent Technologies), followed by paired‐end sequencing on a MiSeq instrument . Bioinformatic analysis was performed using the SureCall software (Agilent Technologies). Common germline polymorphisms reported in public databases were excluded and nonsense, frameshift, splice site, nonsynonymous variants were considered as mutations. Minimal allele frequency for mutation calling was set at 0.3.Target deep sequencing of RAS pathway genes including the 33.1TCF3‐HLF .We prospectively evaluated MRD levels in a t17;19)‐ALL case, treated with a high risk (HR) regimen in the TCCSG L04‐16 study,7;19‐ALL 3.2l‐Asp, and CPM) used in the induction therapy of the HR regimen of the TCCSG L04‐16 study. We determined the IC50 of each agent based on the dose‐response curve in an alamarBlue cell viability assay ‐ALL cell lines (median: 300 ng/mL) is significantly higher than that of t‐ALL cell lines (median: 15 ng/mL) (P = 0.006 in Mann‐Whitney test)   ‐ALL cell lines tended to be higher than that in t‐ALL cell lines ‐ALL cell lines in comparison with t‐ALL cell lines ‐ALL case in comparison with the t‐ALL cases. To verify resistance of t‐ALL to induction therapy in vitro, we analyzed the sensitivities of four t‐ALL cell lines, as well as 16 t‐ALL cell lines, to six agents  Figure B. IC50s ) Figure B and Pre) Figure C in t than those in the DNR‐treated t‐ALL cell lines (22%)  than those in the VCR‐treated t‐ALL cell lines (55%) ‐ALL and seven representative t‐ALL cell lines. When treated with 50 ng/mL of DNR, cell viabilities in two t‐ALL cell lines (UOCB1 and HALO1) and two t‐ALL cell lines (KOPN60 and 697) were 80%, 83%, 6%, and 4%, respectively Figure A. A simi) Figure C. Simila) Figure C.3.3l‐Asp), four  and five  agents ‐ALL cell lines to multiple agents used in induction therapy, we analyzed the combined sensitivities to three ‐ALL and t‐ALL cell lines were closely correlated with that of VCR  , MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP6 (ABCC6), and BCRP (ABCG2). Among nine genes, MRP3, MRP5, and MRP6 were undetectable in BCP‐ALL cell lines. Gene expression levels of ABCB1 were significantly higher in t‐ALL cell lines than in other BCP‐ALL cell lines including the t‐ALL cell lines ‐ALL cell lines than in other BCP‐ALL cell lines, although they were almost equal between the t‐ALL cell lines and t‐ALL cell lines ‐ALL cell lines and other BCP‐ALL cell lines. Since ABCB1 gene expression levels were significantly higher in t‐ALL cell lines, we next analyzed the cell surface expression of P‐gp using flow cytometry. Among four t‐ALL cell lines, P‐gp was clearly detectable in HALO1 and UOCB1 and marginally detectable in Endo‐kun, but almost undetectable in YCUB2  in 32 BCP‐ALL cell lines. Cell surface expression level of P‐gp was relatively higher in t cell lines than in t‐ALL cell lines (P = 0.088 in Mann‐Whitney test)  Figure A. Since s Figure . No sign3.5To test the involvement of P‐gp in resistance to DNR and VCR in t‐ALL cell lines, we evaluated the functional drug‐efflux activity using calcein‐AM (CAM), an ABC transporter‐dependent dye.3.6P = 0.018 in Kaplan‐Meier analysis).We finally tried to confirm the involvement of P‐gp in DNR resistance of t‐ALL in vivo using NSG mice. After inoculation of HALO1 cells into NSG mice, we treated mice with DNR alone or DNR in combination with CyA for 5 days  in t‐ALL and t‐ALL cell lines using a next generation sequencer ‐ALL cell lines, respectively, and gene mutation in RAS pathway was detectable in all t‐ALL cell lines. In contrast, mutations in PTPN11, NRAS, KRAS, and NF1 genes were detectable in none, three, four, and one of 16 t‐ALL cell lines, respectively, and gene mutation in RAS pathway was detectable in seven out of 16 t‐ALL cell lines. Incidence of RAS pathway mutation tended to be higher in t‐ALL cell lines than in t‐ALL cell lines (P = 0.094 in chi‐square test).Association of gene mutation in the RAS pathway with poor therapeutic outcome in childhood ALL is controversial.er Table A. Mutati3.8KRAS mutation are sensitive to inhibitors of MAP kinases in vitro.KRAS mutation. We determined IC50 of selumetinib in t‐ALL and t‐ALL cell lines using an alamarBlue cell viability assay ‐ALL cell lines (two cell lines with KRAS mutation and two cell lines without it) (median: 100 μmol/L) was almost identical to that in 16 t‐ALL cell lines (four cell lines with KRAS mutation and 12 cell lines without it) (97 μmol/L). Of note, six cell lines with KRAS mutation [two t‐ALL cell lines and four t‐ALL cell lines] were relatively more sensitive to selumetinib than 14 cell lines without KRAS mutation [two t‐ALL cell lines and 12 t‐ALL cell lines] (P = 0.060 in Mann‐Whitney test).A recent report revealed that ALL samples with y Figure B. The IC3.9l‐Asp than those without it (P = 0.050 in Mann‐Whitney test). Then, we compared the total score of three, four, and five drug sensitivities of t‐ALL cell lines with RAS pathway mutation with those without it .We finally analyzed a possible association of RAS pathway mutation with drug resistance in t‐ALL and t‐ALL cell lines, since RAS pathway mutation was observed more frequently in t‐ALL cell lines. Association of RAS pathway mutation with sensitivity to each of the five drugs was not statistically significant in t‐ALL cell lines Figure , but t‐ALL during intensified induction therapy. Fischer et al have reported that MRD remains positive at the end of induction therapy in most t‐ALL cases.50s of Pred, VCR, and DNR in four t‐ALL cell lines were significantly higher than those in 16 t‐ALL cell lines. We also confirmed that the combined sensitivities to three , four , and five  agents in t‐ALL cell lines were significantly higher than in t‐ALL cell lines. These observations suggest that resistance to multiple agents, in particular to Pred, VCR, and DNR, may be associated with poor response to induction therapy of t‐ALL.In this study, case of t;19‐ALL d50s of DNR and VCR that are sensitive to ABC transporters are correlated with each other in t‐ALL and t‐ALL cell lines. Consistently, among eight genes of ABC transporters, the gene expression level of ABCB1 was significantly higher in t‐ALL cell lines than in t‐ALL cell lines. We also found that cell surface expression of P‐gp tended to be higher in t‐ALL cell lines than in t‐ALL cell lines, suggesting that P‐gp expression may be involved in resistance to VCR and DNR of P‐gp‐positive t‐ALL cell lines. P‐gp was active in P‐gp‐positive t‐ALL cell lines, as we found that inhibitors for P‐gp such as CyA and nilotinib intensified CAM staining and antileukemic activities of VCR and DNR. More precisely, we confirmed that knockout of P‐gp expression by CRISPR‐Cas9 overcomes resistance to VCR and DNR in a P‐gp‐positive t‐ALL cell line. We additionally confirmed that a combination of CyA with DNR significantly prolonged survival of NSG mice inoculated with a P‐gp‐positive t‐ALL cell line in comparison with DNR alone. Although in vivo combination activities of nilotinib and verapamil with DNR were not directly tested, these findings strongly suggest that overexpression of P‐gp is involved at least partly in resistance to VCR and DNR of P‐gp‐positive t‐ALL cell lines. Baudis et alABCB1 gene expression is detectable by RT‐PCR in Reh cells transfected with TCF3‐HLF under the influence of the zinc‐inducible promoter, suggesting a possibility that the ABCB1 gene is one of the downstream target genes of TCF3‐HLF.To verify the underlying mechanism(s) for poor response of t‐ALL to VCR and DNR, we focused on ABC transporters,NR3C1) gene (specific for GRα and GRγ isoforms), shows significant correlation with IC50 of Pred in 72 BCP‐ALL cell lines.38 However, an association of higher P‐gp expression with resistance to glucocorticoids is unlikely at least in HALO1 cells, since knockout of P‐gp expression by CRISPR‐Cas9 did not overcome Dex resistance.In addition to VCR and DNR, t‐ALL cell lines were significantly more resistant to Pred than t‐ALL cell lines. Sensitivity of ALL cells to glucocorticoids is highly associated with expression of the glucocorticoid receptor (GR). Our previous analyses demonstrated that the gene expression level of GR, analyzed by real‐time RT‐PCR of exons 8 and 9a of GR ‐ALL cell lines and 12 t‐ALL cell lines without the mutation. This higher sensitivity to selumetinib seems to be consistent with the above hypothesis that RAS pathway mutation may provide an advantage for in vitro cell growth and/or cell survival of the cell lines. Furthermore, among 16 t‐ALL cell lines, multidrug resistance was significantly more common in the cell lines with RAS pathway mutation than those without it. These observations suggest that frequent RAS pathway mutation may be involved at least partly in the aggressive clinical course of t‐ALL.A previous report revealed that genes in RAS pathway are frequently mutated in clinical samples of t17;19)‐ALL cases but not in t‐ALL cases.;19‐ALL cIn summary, our observations of a large panel of cell lines revealed that t‐ALL cell lines were significantly more resistant to multiple agents in induction therapy in comparison with t‐ALL cell lines. Although there are some limitations in using the cell lines in drug sensitivity studies, our findings seem to be consistent with the clinical notion that t‐ALL is resistant to intensified induction therapy in comparison with t‐ALL. Thus, these cell lines may be optional tools to study the mechanism(s) for drug resistance and to verify the activities of newly developed compounds in t‐ALL.The authors declare no competing financial interests.AW performed the research, analyzed the data, and wrote the paper; T. Inukai designed the research study, performed the research, analyzed the data, and wrote the paper as a principal investigator; KK, MA, MT, TF, HF, TN, KT, T. Ito, TT, DH, TS, SS, HO, KA, and K.G performed the research; JF, HG, ME, TL, MK, MM, JK T.Inaba, and HT contributed essential samples and cell lines; EI, AO, and KS supervised the project; and all authors contributed to the final draft. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Nepeta crispa essential oil was compared with propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines.Attempts to use the plant products are to be an appropriate option due to substantial concerns about human health and environmental problems of using synthetic pesticides. Therefore, the cytotoxicity of Spodoptera frugiperda (Lepidoptera: Noctuidae) and mouse fibroblast cells, respectively, were obtained from the National Cell Bank of Pasteur Institute of Iran. About a number of 2 × 103 cells were placed into the wells of 96-well plate experiments. Then, appropriate concentrations of essential oil of N. crispa plant and propoxur added to the wells. The cells were allowed to grow for 3-5 days and estimated the numbers of cells. The cells of control experiment wells contained only cells with dimethyl sulfoxide. All control and treatment experiments repeated at least four replicates.The cell lines of Sf9 and L929 which were derived from the ovary glands of fall armyworm, N. crispa essential oil against vertebrate (L929) cell line was gradually decreased in accordance with plant concentrations, while the cytotoxicity of N. crispa essential oil against invertebrate (Sf9) cell line was strongly increased in accordance with plant concentrations.Propoxur had negative effects on the viability of both invertebrate (Sf9) and vertebrate (L929) cell lines. The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased in accordance with propoxur concentrations. The cytotoxicity of N. crispa plant had negative effects on invertebrate cell viability with the differences that the products derived from plants possessing of biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals.Plant essential oil not only had no negative effects but also had boosting effects on vertebrate cell viability. Essential oil of  Attempts to use the plant products as natural pesticides usually based on plant essential oils are considered as a good candidate for the management of arthropod vectors of diseases and agricultural pests due to possessing of readily biodegradable and environmentally friendly derivatives -5. ThereThere are substantial concerns about human health and environmental problems of synthetic pesticides. Pesticides have potential side effects despite their importance to public health. Exposures to various types of insecticides may be a risk factor for cancers. Several undesirable effects of pesticides are maybe toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users ,9-12. InNepeta crispa Willd.  that is popular in Iranian traditional medicine, especially with Hamadan Province people. N. crispa have insecticidal activity with antimicrobial and antifungal properties. The N. crispa plants are autochthonous of Iran climate, especially Hamadan Province [One of the most natural aromatic plants in Iran is Province -22.N. crispa plant, but a simultaneous comparative study about the cytotoxicity of essential oil of N. crispa with synthetic pesticides such as propoxur against the cell lines of invertebrates and vertebrates would be a particular of importance. Therefore, in this study, we compared the cytotoxicity of N. crispa essential oil with propoxur against invertebrates and vertebrates cell lines.Many studies have been conducted on the insecticidal properties of The study was approved by the Ethics Committee of Hamadan University of Medical Sciences, Hamadan, Iran, with approval number: IR.UMSHA.REC.1396.320.Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae). Cell line of Sf9 is routinely cultured and maintained at 27°C in 5 ml of Grace’s insect cell culture medium in 25 cm2 culture flasks, enriched with 10% fetal bovine serum at Pasteur Institute of Iran. The doubling time of Sf9 cells was found to be 18-24 h under optimum conditions. Sf9 invertebrate cells were subcultured every 3 days. Cell line of L929 was derived from mouse fibroblast cells. It was maintained at 37°C in 3 ml of Dulbecco’s Modified Eagle Medium Media (Gibco®) in 25 cm2 culture flasks, enriched with 10% fetal bovine serum, and buffered with 4% sodium bicarbonate in an atmosphere of 5% carbon dioxide. The doubling time of L929 cells for cultures was approximately 24 h and the cell was subcultured every 6 day [Invertebrate (Sf9) and vertebrate (L929) cell lines were obtained from the National Cell Bank of Pasteur Institute of Iran. Cell line of Sf9 derived from the ovary glands of fall armyworm, ry 6 day .N. crispa  during their flowering stage were collected from Avicenna Medicinal Herbs Research Center, Hamadan Province of Iran in June 2017. The plant was confirmed by a voucher specimen (no. 72) in the Department of Pharmacognosy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran. A total of 1000 g powder of shade-dried aerial parts of N. crispa were subjected to hydrodistillation using a Clevenger-type apparatus for 4 h. The essential oil was dehydrated over anhydrous sodium sulfate and transferred into amber-colored vials to store in a refrigerator at 4°C for further work.Technical grade of propoxur  was purchased from Guangdong Company, China (Mainland). The aerial parts (foliage) of N. crispa plant was dissolved with 1 ml of dimethyl sulfoxide (DMSO) due to hydrophobic properties and then diluted with sterilized distilled water to prepare the concentrations of 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, and 10−3 ng/μL containing 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng/μL, respectively. Propoxur technical grade (97%) was dissolved with ethanol 96% to prepare the concentrations of 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, and 10−3 µg/μL containing 0.000097, 0.00097, 0.0097, 0.097, 0.97, 9.7, 97, and 970 µg/μL, respectively.Essential oil (0.1 mg) of N. crispa plant and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, about 2 × 103 cells/100 µl of culture medium were placed into the wells of a 96-well plate of treatment experiments and then appropriate concentrations of N. crispa essential oil and propoxur added to the wells. We allowed the cells to grow for 3-5 days and estimated the number of cells as described. Control cell wells contained only cells with 1µl/mL of DMSO. All treatment and control experiments repeated at least four replicates.To consider the cytotoxicity of essential oil of in vitro cytotoxicity which measures the active metabolism of the cells. In this coloring solution, dehydrogenase enzyme reduced the MTT and produced blue formazan.The base method for cell estimation is the Mossman method, which uses 3--2,5-difenyltetrazolium bromide . MTT is a quantitative coloring for living cells and cell proliferation, and it is a known method for The wells of 96-well plates containing invertebrate (Sf9) and vertebrate (L929) cell lines were incubated with 10 µ MTT for 3 h at 36°C and 27°C, respectively. After the blue formazan and cells settled out and the supernatant was removed, 100 µ of DMSO was added to any well of 96-well plate, shaked for 15 min and then the absorbance of the solution read at 492 nm using ELISA reader .N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines were estimated by Microsoft Excel version 2013. The trend lines were drawn by clicking on graph line distribution and selecting “add trendline” option using procedure described in previous studies [N. crispa plant and propoxur between control and treatments, and cross treatments of N. crispa plant and propoxur. p<0.05 was considered statistically significant.The cytotoxicity trend lines of  studies -28.The eN. crispa essential oil and propoxur cytotoxicity against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. Figures-N. crispa essential oil and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines, respectively. N. crispa essential oil (ng/μL) and propoxur (μg/μL) against invertebrate (Sf9) and vertebrate (L929) cell lines. N. crispa plant, and propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines.Tables-ebrate L99 cell liN. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant from 10−10 to 10−3 µg/μL  cell line was gradually decreased with a moderately increasing slope in accordance with essential oil concentrations of N. crispa plant from 10−10 to 10−3 µg/μL  cell line with control (p<0.05)  cell line with control (p=0.068)  cell line with control  cell line with control (p>0.05)  . Wilcoxop=0.068) , even th control . Wilcoxo(p>0.05) , even the of Sf9 .−10 to 10−3 µg/μL  cell line with control (p>0.05) (−10 and 10−5 to 10−3 concentrations of propoxur against invertebrate (Sf9) cell line (−10 and 10−3 concentrations of propoxur against vertebrate (L929) cell line with control (p<0.05) (The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased with a relatively low decreasing slope in accordance with propoxur concentrations from 10 Figures-, b and 3(p>0.05) , even thell line . There w(p<0.05) .N. crispa plant against invertebrate (Sf9) cell line was strongly increased with an intensive increasing slope in accordance with essential oil concentrations of N. crispa plant, while the cytotoxicity of essential oil of N. crispa plant against vertebrate (L929) cell line was gradually decreased with a moderately increasing slope in accordance with essential oil concentrations of N. crispa plant. The cytotoxicity of propoxur against invertebrate (Sf9) and vertebrate (L929) cell lines was gradually increased with a relatively low decreasing slope in accordance with propoxur concentrations  and vertebrate (L929) cell lines, and invertebrate (Sf9) cell line, respectively (p>0.05), even though there were significant differences between cross treatments (10−10–10−4) of N. crispa plant and propoxur concentrations against vertebrate (L929) cell line (p<0.05) (Wilcoxon signed-rank test did not show significant differences between separatel(p<0.05) .−5-10−3 and 10−8-10−3 concentrations of propoxur with control group against the invertebrate (Sf9) and vertebrate (L929) cell lines, respectively, at p<0.05 or p<0.001 levels  and vertebrate L929) cell lines. These results confirm the reports of the previous studies that concluded insecticides decreased growth of the invertebrate and vertebrate cell lines  cell lin. Compare1 levels .N. crispa plant did not have negative effects on the viability of vertebrate (L929) cell line. Compared with the control group, the highest rate of cell viability was observed in the experiment treatments which were treated with essential oil of N. crispa plant. The viability of vertebrate (L929) cell line which was exposed to different essential oil concentrations of N. crispa plant was higher than control group and increased with increasing essential oil concentrations of N. crispa plant. The boosting effects of experiments of N. crispa plant on the viability of vertebrate (L929) cell line were also confirmed by Wilcoxon signed-rank test by observing significant differences between the treatments of 10−10 and 10−3 essential oil concentrations of N. crispa plant with propoxur treatments of 10−10-10−3 concentrations and control group against the vertebrate (L929) cell line at p<0.05 or p<0.001 levels. In addition to some previous benefits of the natural products deriving from natural plants [N. crispa plant on the viability of cell line of vertebrates may be considered as the new benefits of the products deriving from natural plants.Unlike propoxur, essential oil of l plants ,32, thisN. crispa plant had negative effects on the viability of invertebrate (Sf9) cell lines with the differences that the products deriving from natural plants possessing of readily biodegradable and environmentally friendly derivatives, hydrolyzing rapidly in nature, and nearly having no destructive effects on environment, humans, or the mammals [N. crispa plant (ng/μL) was 1000 folds lower than concentrations of propoxur (μg/μL). The viability of invertebrate (Sf9) cell lines which were exposed to essential oil concentrations of 10−9 and 10−7-10−3N. crispa plant was lower than control group and decreased with increasing essential oil concentrations of N. crispa plant.Like propoxur, essential oil of  mammals ,2. In adN. crispa plant on the viability of invertebrate (Sf9) cell lines were also confirmed by Wilcoxon signed-rank test by observing a significant difference between the treatments of 10−7 and 10−3 essential oil concentrations of N. crispa plant with control group against the invertebrate (Sf9) cell lines at p<0.001 level. In addition to that, the food chain and ecosystem contamination with pesticides, non-target organism extinction, insecticide resistance of insects, mutation, and pollution of water and soil have been critical problems using pesticides in the past decades. However, with the application of the products deriving from natural plants, there is no concern about their potential side effects such as toxicity to non-target organisms, residues of pesticides, depletion of ozone layer, environmental pollution, and direct toxicity to users. We will not encounter probably some psychotic disorders, neurological diseases and fatigue of muscles or cancers, and face up to severe insecticide effects on performance of vertebrate reproductive system [The negative effects of essential oil of e system ,9-12. Soe system -19.N. crispa plant did not have negative effects on the viability of vertebrate cell line. The viability of vertebrate cell line was increased with increasing essential oil concentrations of N. crispa plant. This boosting effect of N. crispa plant on the viability of vertebrate cell line may be considered as the new benefits of the products deriving from natural plants.Based on the results of the study, propoxur had negative effects on the viability of both invertebrate and vertebrate cell lines. Unlike propoxur, essential oil of AS and AZ planned the project. MB and HN conducted the experimental laboratory work and analyzed the data. SS assisted in analyzing of experimental data. AS and HN wrote the manuscript. AS supervised the project. All authors discussed the results and implications and commented on the manuscript at all stages. All authors read and approved the final manuscript.


Mesua beccariana was carried out. A new cyclodione, mesuadione (1), along with several known constituents which are beccamarin (2), 2,5-dihydroxy-1,3,4-trimethoxy anthraquinone (3), 4-methoxy-1,3,5-trihydroxyanthraquinone (4), betulinic acid (5) and stigmasterol (6) were obtained from this ongoing research. Structures of these compounds were elucidated by extensive spectroscopic methods, including 1D and 2D-NMR, GC-MS, IR and UV techniques. Preliminary tests of the in vitro cytotoxic activities of all the isolated metabolites against a panel of human cancer cell lines Raji (lymphoma), SNU-1 (gastric carcinoma), K562 (erythroleukemia cells), LS-174T , HeLa , SK-MEL-28 , NCI-H23 (lung adenocarcinoma), IMR-32 (neuroblastoma) and Hep-G2 (hepatocellular liver carcinoma) were carried out using an MTT assay. Mesuadione (1), beccamarin (2), betulinic acid (5) and stigmasterol (6) displayed strong inhibition of Raji cell proliferation, while the proliferation rate of SK-MEL-28 and HeLa were strongly inhibited by stigmasterol (6) and beccamarin (2), indicating these secondary metabolites could be anti-cancer lead compounds in drug discovery.An investigation on biologically active secondary metabolites from the stem bark of  Mesua, which is from the Clusiaceae family is a prolific producer of metabolites. Different parts of Mesua species are traditionally used as folk medicine for treatment of dyspepsia, fever, renal diseases and even as a poultice  (12), 136 (12), 123 (20), 109 (33), 96 (82), 82 (100), 67 (80), 56 (10). For 1H- and 13C-NMR spectra, see Beccamarin (2). Yellow solid. m.p. 139.0–139.6 °C. UV (EtOH) λmax nm (log ε): 209, 229, 281 and 348. IR νmax (cm−1): 3400, 2971, 1741, 1605 and 1466. MS m/z (rel. int.): 406, 392, 377, 350, 349, 293, 43. The 1H and 13C-NMR (CDCl3) spectral data are consistent with published data [2,5-Dihydroxy-1,3,4-trimethoxyanthraquinone (3). Orange solid. UV (MeOH) λmax nm (log ε): 218, 276 and 410. IR νmax (cm−1): 3400, 2920, 2840, 1660, 1630, 1540. MS m/z (rel. int.): 330, 315, 312, 297, 287, 272, 227, 58. The 1H and 13C-NMR (CDCl3) spectral data are consistent with published data [4-Methoxy-1,3,5-trihydroxyanthraquinone (4). Orange solid. UV (MeOH) λmax nm (log ε): 279, 320, 425, 470 and 485. IR νmax (cm−1): 3420, 2920, 2860, 1720, 1630, 1470. MS m/z (rel. int.): 286, 268, 257, 243, 212, 180. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [hed data .Betulinic acid (5). White solid. m.p. 290–291 °C. IR νmax (cm−1): 2939, 1687, 1453, 1375. MS m/z (rel. int.): 456, 248, 207, 189, 121, 107, 95, 81, 69, 55. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [Stigmasterol (6). White needles. m.p. 165–166 °C. IR νmax (cm−1): 3399, 2939, 1457, 1374. MS m/z (rel. int.): 412, 255, 159, 145, 95, 81, 69, 55. The 1H and 13C-NMR (CDCl3) spectral data are consistent with the published data [2 T-flask and maintained at 37 °C in 5% CO2 humidified incubator. All cell lines were sub-cultured depending on the confluence of cells and observed under an inverted microscope for cell viability and checking for any contamination. Once the cells reach the confluent state, cell numbers were evaluated using the trypan blue staining method in a hemocytometer. All cell lines were deposited in American Type Culture Collection (ATCC). Besides, all the cell lines except for the Raji and HeLa were classified under the biosafety level 1 by ATCC.Nine cancer cell lines: Raji, SNU-1, LS-174T, HeLa, SK-MEL-28, NCI-H23, IMR-32, Hep-G2 and K562 were obtained from Universiti Tunku Abdul Rahman (UTAR). The Raji, SNU-1, K562, HeLa, Hep G2 and NCI-H23 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) while the LS-174T, SK-MEL-28 and IMR-32 were maintained in MEM supplemented with 10% FBS. All the cell lines were cultured in 75 cmThe cytotoxicity study was performed by the MTT assay as described by Mosmann . SelecteAn aliquot of 100 µL of each sub-stock with different concentrations were added to each well together with 100 µL of selected cells to give concentrations of 100.00, 50.00, 25.00, 12.50, 6.25 and 3.13 µg/mL and made up to a final volume of 200 µL in each well. Cells with no extracts  and 200 µL of medium only (blank medium—negative control) were prepared in the same plate. Samples and controls were prepared in triplicate.2 incubator. After 72 h, 20 µL of MTT solution was added to all the wells and incubated for 3 h in a 5% CO2 incubator. The plate was then spun at 3,000 rpm for 10 min. Supernatant from each well (160 µL) was discarded and then DMSO (160 µL) was added to dissolve the purple formazan crystals.The plate was then incubated for 72 h at 37 °C in 5% COThe absorbance of each well was determined using a microplate reader at 550 nm. The average absorbance of each crude extract was calculated and the average value was used to determine the percentage of cell viability by using the following formula:where: A = absorbance of sample       B = absorbance of negative control       C = absorbance of positive control50 values were obtained from the plotted graph. Further dilutions were only performed on the compounds with IC50 values less than 3.13 µg/mL. Three independent experiments were conducted to assure the accuracy of the results. Kaempferol and quercetin were used as standard compounds throughout the cytotoxicity experiments.A graph of percentage of cell viability versus concentration was plotted for each extract and pure compound. The IC1), was isolated from the stem bark of Mesua beccariana along with several known constituents 2–6. Compounds 1, 2, 5, and 6 were strongly cytotoxic towards the Raji cell line. These phytochemical and cytotoxicity investigations which demonstrated the cytotoxic effects of these compounds suggest them to be potential chemopreventive agents in drug discovery. A new cyclodione, mesuadione (


Acaromyces ingoldii FS121, which was obtained from the China South Sea, yielded a new naphtha-pyrandione analogue, acaromycin A (1) and a new thiazole analogue, acaromyester A (2), as well as the known compound (+)-cryptosporin (3). Their structures, including absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) spectra. Compounds 1–3 were evaluated for in vitro growth inhibitory activities against four tumor cell lines , wherein compounds 1 and 3 exhibited considerable growth inhibitory effects, with IC50 values less than 10 µM.Activity-guided isolation of the fermentation broth of the deep-sea derived fungus  Marine organisms live in a biologically competitive environment with unique environmental conditions, therefore marine-derived fungi have been proven to be a rich source of structurally unique and biologically active secondary metabolites ,7,8,9,10Acaromyces ingoldii FS121 showed potent cell growth inhibitory effects against SF-268, MCF-7, NCI-H460 and HepG-2 human tumor cell lines , five sp2 quaternary carbons δC 162.2 (C-7), 155.2 (C-10a), 132.1 (C-9a), 118.8 (C-4a), 114.1 (C-5a), three sp2 methines δC 137.2 (C-6), 124.7 (C-8), 119.3 (C-9), three sp3 methines δC 76.2 (C-2), 66.9 (C-3), 65.1 (C-4), and one methyl δC 16.7 (C-11). As six of nine degrees of unsaturation account for four double bonds (eight sp2 carbons), and two carbonyl groups, the remaining degrees of unsaturation requires that 1 be a tricyclic system. The HMBC correlations (H 11.68) to C-5a, C-7 and C-8. Furthermore, the 1H-1H COSY spectrum -cryptosporin (3), except for the OH at C-9 in 3 was locked at C-7 in 1. Therefore, the planar structure of 1 was confirmed.Acaromycin A , H-3 , H-4 ] with those of 3 . This indicated that these protons were cofacial and designated as β-oriented, whereas the Me-11 was α-oriented in 1. The absolute configuration of 1 was determined by the CD spectrum. The absolute configuration of (+)-cryptosporin (3) was established by synthesis and its CD spectra data was reported in the literature . This assumption was further confirmed by the HMBC correlations from H-2 to C-1, C-3 and C-8, H-5 to C-3 and C-7, and H-8 to C-6 and C-4. Besides, the 1H-NMR of 2 also exhibited a methyl signal at δH 2.24 , two methylene protons at δH 3.02  and 3.84 , and an olefinic proton due to the thiazole ring at δH 8.61 , suggesting the presence of a 4-methyl-5-thiazoleethanol substructure in 2, which has been reported in the literature + 299.0533 .Acaromyester A (2): light pink oil; UV (MeOH) λmax (log ε) 202 (4.24), 225 (4.08), 277 (3.44) nm; IR νmax 3309, 3178, 1732, 1716, 1516, 1236, 1145, 804, 783 cm−1; 1H- and 13C-NMR, see m/z [M + H]+ 278.0845 .1–3 were tested against four tumor cell lines, including MCF-7 (human breast adenocarcinoma cell line), NCI-H460  and SF-268 (human glioma cell line), HepG-2 (human hepatomacarcinoma cell line) by the SRB method pyran-5,10-quinone and a new heterodimeric compound combining a hydroxyphenylacetate and a 4-methyl-5-thiazoleethanol substructure, which were isolated from the fermentation broth of Acaromyces ingoldii FS121. It merits attention that compounds 1 and 3 contained a 3,4-dihydro-2H-naphtho-pyran-5,10-quinone core framework, which exhibited a wide range of biological activities [1–3 were evaluated, wherein compound 1 and 3 showed considerable activities against MCF-7, NCI-H460, SF-268 and HepG-2 tumor cell lines.In summary, we have described herein the isolation and structural elucidation of a new 3,4-dihydro-2


Byrsonima crassifolia (L.) Kunth and B. verbascifolia (L.) DC.) and tapereba (Spondias mombin) are Amazonian fruits that contain bioactive compounds. Biochemical and molecular characterization of these fruits can reveal their potential use in preventing diseases, including cancer. The extracts were characterized regarding the presence and profile of carotenoids by high-performance liquid chromatography (HPLC), total phenolic content by the Folin–Ciocalteu assay, and antioxidant activity by antioxidant value 2,2-diphenyl-1-picrylhydrazyl (DPPH) content analysis, 22,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) content analysis, Ferric-Reducing Ability of Plasma (FRAP), and Oxygen Radical Absorbance Capacity (ORAC) analysis. The extracts of tapereba and murici studied were important sources of total carotenoids and lutein, respectively. The extracts were then tested for their effect on the viability of the A2780 ovarian cancer (OC) cell line and its cisplatin (CDDP)-resistant derived cell line, called ACRP, by using MTT -2,5-diphenyltetrazolium bromide) assays. Their influence on cell cycle and apoptosis were analyzed by using flow cytometry. Murici and tapereba cell extracts exhibited a strong bioactivity by inhibiting A2780 and ACRP cell viability by 76.37% and 78.37%, respectively, besides modulating the cell cycle and inducing apoptotic cell death. Our results open new perspectives for the development of innovative therapeutic strategies using these Amazon fruit extracts to sensitize ovarian cancer cells to current chemotherapeutic options.Murici ( Ovarian carcinoma (OC) is a highly heterogeneous disease and despite advances in understanding it, patients suffering from this disease still have poor prognostic rates related to late diagnosis and rapid progression . In addiAlternative approaches or combination therapies including CDDP could be important options for the treatment of OC. It is known that Brazil has the greatest biodiversity in the world, with many species still unknown or little studied. Fruit species have high economic values, both in the fresh fruit trade and use as raw materials for agroindustry . The AmaAmong the main preventive strategies for health is chemoprevention, which consists of the use of natural or synthetic chemical agents to prevent, interrupt, stabilize, or reverse the genesis of cancer ,7. Some These fruits are endemic in some ecosystems in Brazil, but produced predominantly in the Amazon region in forest yards and are then harvested by extractivism. They present an increasing economic importance in tropical regions, especially in the field of eccentric juices and desert markets ,13. The Impressive progress is being made in discovering the role of bioactive compounds in reducing the risk of cancer and the underlying biological mechanisms that account for these effects. Epidemiological studies suggest that cancer risk is related to dietary intake of fruits and vegetables that are rich in carotenoids and phenolic compounds . These bME has been described as a good source of lutein and zeaxanthin . BesidesTAP is also a source of phenolic compounds having tannins as the main component. Its antioxidant capacity may also be related to the presence of compounds such as vitamin C and carotenoids. Large amounts of gallic acid and quercetin have been found in TAP, and its pulp can effectively inhibit oxidation, which is attributed to its yellow flavonoids, carotenoids, and chlorophyll ,16,17.Based on the need to develop more effective treatment strategies to reverse chemoresistance and the survival rates in OC patients and the potential properties of ME and TAP as sources of bioactive compounds, this paper investigates the use of extracts from these Amazonian fruits as auxiliary tools to modulate the cell viability and survival of A2780 and ACRP OC cell lines.Pulps of ME and TAP packaged in sealed and labeled plastic bags (1 kg) were supplied by a company from Pará  and stored at a controlled temperature (−18 °C). The frozen pulp was transported in an ice chest containing dry ice to the Laboratory for Analysis of Functional Foods (LAAF-UNIRIO), Rio de Janeiro (Brazil), where they remained frozen (−18 °C) until the moment of analysis.® LD 300, São Carlos, SP, Brazil) for 24 h. After this process, extracts were frozen at −20 °C until use in the experiments [Approximately 250 g of pulp of ME and TAP was extracted with 80 mL of distilled water and then shaken for 2 h. After the pulp maceration period, the aqueous ME and TAP extracts were filtered through Whatman #1 filter paper. The extracts were then frozen at −80 °C in an ultra-freezer and lyophilized  according to Rodriguez-Amaya (2001) [®, Milford, KS, USA), with 80% MeOH , and 20% methyl t-butyl ether as the mobile phase with a column temperature of 33 °C. β-Cryptoxanthin, lutein, zeinoxanthin, α-carotene, β-carotene, and zeaxanthin were quantified.The composition and level of carotenoids in extracts of powdered ME and TAP pulps were determined using high-performance liquid chromatography  . Saponif® 340, Haverhill, MA, USA) in triplicate and the results were expressed as mg gallic acid equivalent (GAE)/mL extract.The Folin–Ciocalteu assay was performed to determine the concentration of total phenolics in the ME and TAP aqueous extract powders. The method was performed as described by Singleton and Rossi (1999) . The extThe antioxidant capacity of the ME and TAP extracts was measured by the ORAC assay, according to Prior and Hoang (2000) . PBS  , by the A reaction mixture of 2.5 mL DPPH methanolic solution (0.06 mM) with 0.5 mL of ME or TAP extracts was mixed completely and left in the dark at a controlled temperature for 60 min. The absorbance of the blend was measured with a spectrophotometer (Turner 340) at 515 nm in triplicate .TEAC assay was performed following the procedure proposed by Rufino et al. (2007) . The ABT2 atmosphere. Stock cultures in flasks were grown to 80% confluence and routinely subcultured. Cell morphology was observed using a Zeiss Observer Z1 microscope and all images were captured using Axio-Vision Rel. 4.8 software . For each experiment, the cells were seeded at 2 × 105 cells/cm2 and 5 × 105 cells/cm2 densities in 96-well plates and 6-well plates.The human ovarian carcinoma cell line (A2780) was obtained from the National Cancer Institute  and the CDDP-resistant cell line (ACRP) was developed using a pulse selection strategy followed by recovery in free media of drugs to mimic the clinical administration of chemotherapy. The two cell lines (A2780 and ACRP) were routinely maintained in an RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin, under 5% CO5 in 96-well plates in triplicate and incubated for 24 h following the procedure for cell adhesion. Then the medium was removed and the cells were exposed to nine concentrations of both the extracts (0.01 to 20 mg/mL) for 24 h. The treatment with fruit extracts in combination with CDDP, previously defined at 10 µM and 80 µM, was set for the A2780 and ACRP cell lines, respectively [The cytotoxic effects of ME and TAP extracts were monitored by the 3--2,5-diphenyltetrazolium bromide (MTT) assay. A2780 and ACRP cells were seeded at 2 × 10ectively , while f5 cells/cm2 in a 6-well plate. Cells received treatment with ME and TAP extracts at two concentrations (5 and 20 mg/mL). After incubation for 24 h, the cells were briefly washed with PBS solution and resuspended in 500 μL of Vindelov’s solution containing 0.1% Triton X-100, 0.1% citrate buffer, 0.1 mg/mL RNAse, and 50 mg/mL propionate iodide  [® software .Human ovarian cancer cell lines (A2780 and ACRP) were seeded at 5 × 10MO, USA)  and left® software .The ovarian cancer cell lines were seeded in 6-well plates at concentrations similar to those used for cell cycle analysis. After 24 h of treatment with the extracts at concentrations of 5 and 20 mg/mL, the cells were washed with buffered saline solution (PBS), resuspended in a binding buffer with 5 μL of annexin V FITC and 5 μL of propidium iodide (PI) . Apoptotic and necrotic cells were labeled and quantified in a BD Accuri ™ C6 flow cytometer and analyzed with the CFlown = 9). Data were analyzed using GraphPad Prism . A univariate analysis of variance (ANOVA) and the Tukey’s post-test at a 95% confidence level were used to test cell viability, cell cycle, and apoptosis rates.The results presented are the mean and corresponding standard deviations of three independent experiments performed in triplicate  (1020.39 ± 88.43 μM TE/g), ferric-reducing ability of plasma (FRAP) (1014.71 ± 2.08 μmol ferrous sulfate/g), and Trolox equivalent antioxidant capacity (TEAC) (1620.95 ± 114.65 μmol TE/g), when compared with TAP, which presented mean values of 623.72 ± 38.75 μM TE/g (ORAC), 644.55 ± 10.89 μmol ferrous sulfate/g (FRAP), 78.70 ± 0.28% reduction in  free radicals (DPPH), and 1090.90 ± 296.04 μmol TE/g (TEAC). On the other hand, TAP presented a higher reduction activity in the DPPH assay compared with the ME extract . These vME contained higher levels of total phenolic compounds /mL) compared with TAP (1049.09 ± 95.68 mg GAE/mL), which might explain the antioxidant potential of this extract. Almeida et al. [In other studies, approximately 19 polyphenolic compounds have been identified in murici, including galotannins, quinic acid gallates, proanthocyanidins, quercetin derivatives, and gallium derivatives—substances rarely found in fruits ,28. AuthTAP presented higher levels of total carotenoids (185.92 ± 12.86 μg/g) compared with ME (86.30 ± 8.82 μg/g). Matietto et al.  found toIn the present study, six carotenoids were identified in both the pulps: β-cryptoxanthin, lutein, zeinoxanthin, α- and β-carotene, and zeaxanthin. Among these carotenoids, β-cryptoxanthin, α-carotene, and β-carotene have pro-vitamin A activity . β-cryptCarotenoids are a group of pigments widely distributed in nature that occur in large quantities. These compounds are known for their structural diversity and various biological functions . In fruiTiburshi et al.  found hip < 0.05) (p < 0.05) in A2780 (69.40%) and ACRP (65.54%) cell viability (p > 0.05). When tested at a concentration of 5 mg/mL, TAP promoted a mean reduction of 58.22% in A2780 cell viability. However, it did not promote reduction in A2780 cell viability at the concentrations of 5 and 10 mg/mL compared to ACRP cells, as seen in In order to evaluate the effect of ME and TAP on the viability of the two ovarian cancer cell lines, A2780 and its CDDP-resistant derivative ACRP, these were treated with 0.001–20 mg/mL of each extract for 24 h. ME at a concentration of 20 mg/mL promoted 77.38% and 83.94% decreases in A2780 and ACRP cell viability, respectively, compared with the control group ( < 0.05) . At the iability . MTT tesPessoa et al.  in theirp < 0.05).ME promoted a greater reduction of cell viability in relation to the control than the TAP extract. This decline was also higher in the parental ovarian cancer cell line (A2780) than in the ACRP resistant cell line  and TAP (68.80 ± 5.82%) in A2780 parental cells. The same effect was observed for ME (19.03 ± 3.09%) and TAP (36.60 ± 11.03%) in ACRP, the CDDP-resistant cell line. A decrease of cells in the S and G2/M phases in response to cell treatment with both the extracts was also observed (p < 0.05). The highest concentration of cells in the G0/G1 phase was observed in response to cell treatment with ME in the two cell lines studied (p < 0.05), indicating cell growth arrest of A2780 and ACRP after treatment  and resistant cells (ACRP) demonstrated that the effective dose of CDDP treatment in these situations ranges from 10 µM for A2780 to 80 µM for ACRP cell lines . Based oA2780 cells showed a greater reduction in cell viability after 24 h of treatment with ME and TAP extracts at the concentration of 20 mg/mL. A2780 cells incubated with the lowest concentration of the ME extract (5 mg/mL), as well as those treated with 10 μM of CDDP and a combination of CDDP10 and ME5, presented a similar reduction in cell viability. The treatments combining CDDP10 and TAP extract at the two selected concentrations (5 and 20 mg/mL) showed a similar reduction .Regarding the ACRP cell line, the treatments with the ME and TAP extracts at 20 mg/mL and the treatment combining CDDP80 plus extracts of 20 mg/mL were similar and presented a greater reduction. The treatments with both the extracts at the concentration of 5 mg/mL and combined treatment with CDDP80 and 5 mg/mL ME and TAP extracts were similar to the chemotherapy treatment tested at the concentration of 80 µM. This suggests a preponderant effect of ME and TAP extracts compared to the use of chemotherapy or their concomitant use as a way to promote better results from the synergistic effect of the compounds, especially in cases of resistance to chemotherapy. Similar studies considering fruits from the Amazon region and combined treatment with chemotherapeutic agents in order to inhibit cell viability were not found in the literature.The results indicate that the important bioactive potential of ME and TAP fruits is related to their carotenoid and phenolic content. The levels were higher in the murici extracts, which presented greater action in the cell lines studied, especially in resistance to chemotherapy.From the results obtained, we can conclude that the two Amazonian fruits, ME and TAP, have cytotoxic effects on ovarian cancer cells, leading to cell cycle arrest and apoptosis of diseased cells. The effects of these extracts may open perspectives to design combined approaches to sensitize ovarian cancer cells to current chemotherapeutic drugs such as cisplatin. Therefore, further studies are needed to clarify the putative therapeutic potential of these fruit extracts in ovarian cancer cells.To date, studies reported in the literature about ME and TAP fruit extracts are still scarce, especially of their influence on cancer cell lines. Our study is the first to provide experimental evidence that ME and TAP fruit extracts can inhibit cell viability of parental and CDDP-resistant ovarian cancer cells. The reported studies with these fruit extracts on other cell lines have demonstrated that they are potent antioxidants, providing early evidence that can be used for the development of new chemotherapeutic strategies aiming to prevent the development of several diseases, including cancer.


TRAF1, TNFRSF12A and NFKB2. Our results point to a role of temporal alternative splicing in isoform production, which may alter the outcome of the TNF pathway and impact on tumorigenesis.Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages  compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes  Alternative splicing (AS) belongs to one of the key biological processes, which regulates gene expression diversity and function in eukaryotes. In this process, different coding regions of the pre-mRNA are included in the mature mRNA in a combinatorial way. This leads to exon/intron inclusion or exclusion events in the final transcript form of a gene. As a result, AS is able to generate a myriad of functional proteins from the same gene. This affects cellular functions and pathways, and provides the organism an evolutionary advantage to adapt to its changing environment. Remarkably, it was estimated that around 95% of human genes are alternatively spliced ,2. MoreoIn cancer, changes of AS are well studied, and a deregulated AS is reported as one of the hallmarks of cancer ,7. SeverSeveral studies have illustrated a cancer-specific pattern of AS in various cancer types , prostate, colon and lung cancers) ,13,14,15Per1 and CLOCK) [Increasing evidence points to the regulation of several post-transcriptional processes in a time- dependent manner. Genes and proteins hallmarked by circadian (ca. 24 h period) and ultradian (between 8–12 h period) expression were shown to be conserved across several species and regulate several pathways . These id CLOCK) ,19,20. AOne of the pathways for which published data points to a role of AS in the regulation of elements of the signalling cascade is the TNF (tumour necrosis factor) pathway . As demoDespite the increasing relevance of the time-dependent regulation of cellular processes in cancer progression, the impact of circadian regulation in splicing remains poorly understood. Thus, it is of great biological and clinical relevance to identify timely regulated AS events among elements of the TNF pathway in cancer cellular models. Yet, additional studies at the transcriptome level using circadian data sets are still rare and are essential for the investigation of time-dependent AS regulation ,28. In this work, we characterized the extent of a time-dependent isoform generation in elements within the TNF pathway. We generated 33 time-course data sets (sampling every three hours between ZT12 h and ZT42 h) to investigate the time-dependent production of isoforms in two HL cell lines at different stages  compared to a B lymphoblastoid cell line (LCL-HO) with a focus on TNF pathway-related elements. ZT (zeitgeber time) is defined as the time (in hours) after cell synchronization with medium change. Using a computational approach, we identified several genes involved in the TNF pathway to undergo a time-dependent change in isoform expression with potential implications on the output of the pathway. These include TNF receptor-associated factor 1 (TRAF1), TNF receptor superfamily 12A (TNFRSF12A) and the nuclear factor kappa B subunit 2 (NFKB2) with relevant functions in cell proliferation, migration and apoptosis. Our results further emphasize the potential impact of AS in cellular physiology, in a cancer context, and highlight the effect of temporal isoform switching events as mediators of these processes.p-value cut-off of 0.001, and a difference cut-off of 1 as suggested in the TSIS package  [Previous research pointed at the relevance of splicing in cancer, and identified cell-specific AS changes in HL cell lines ,13,29. Idetails) . The difdetails)  for codiWe further investigated the switches between isoforms in time, focusing in the region around the STPs, and tested for potential functional properties of the resulting transcripts at the sequence level . Such prp-value = 8.360 × 10−7) as well as NMD (adjusted (BH) p-value = 1.870 × 10−3) (VEGFA) are involved in a variety of cancers including skin, pancreatic and colon cancer, as well as lung disease according to our enrichment analysis, which is in agreement with previous studies for the mentioned gene [p-value = 7.099 × 10−8). The SLIT/ROBO pathway has important roles in tumorigenesis, cancer progression and metastasis and thus acts as a regulator for multiple oncogenic signalling pathways including the CD20, mTOR , VEGF and EGFR  pathways [p-value = 6.918 × 10−3), interestingly only in the genes with switches that are unique to this cell line  signalling pathway, which is associated with disrupted organogenesis leading to cancer [p-value = 4.542 × 10−2), the SREBP (sterol regulatory element-binding proteins) pathway  and the circadian clock pathway  to be enriched in the L-1236 cell line  . Additio × 10−3) . The genned gene . Howeverpathways . The ROBo cancer . Additioell line . SREBP bell line . For theell line .HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1) shows an absolute difference of expression of 701.44 TPM between the isoforms ENST00000262584.7 and ENST00000526668.5. JCHAIN (joining chain of multimeric IgA and IgM) shows an absolute difference in expression of 107.69 TPM between the isoforms ENST00000510437.5 and ENST00000543780.5 and NME2 (NME/NM23 nucleoside diphosphate kinase 2) shows an absolute difference in expression of 408.09 TPM between the isoforms ENST00000513177.5 and ENST00000512737.5. The HNRNPH1 gene is involved in splicing. It is overexpressed in many cancer types  and is required for cancer growth and development [HNRNPH1 is different over time for the three investigated cell lines  [MYC gene and its isoforms, which are either differentially or ubiquitously expressed [We computed the difference in expression between the switching isoforms with the TSIS package  and geneelopment ,36,37. Nll lines . There i cancer) . NME2 isxpressed . CD44 gene shows an absolute difference in expression between the isoforms ENST00000263398.10 and ENST00000352818.8 of 164.95 TPM. The TNFRSF12A gene shows an absolute difference in expression of 211.08 TPM for the isoforms ENST00000326577.8 and ENST00000341627.5 and the HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1) gene (involved in splicing and mRNA transport) shows an absolute difference in expression of 171 TPM for the isoforms ENST00000340913.10 and ENST00000547566.5. CD44 encodes for a cell-surface glycoprotein and can act both as a growth- and invasiveness-promoting molecule, and as a tumour-suppressing cofactor via its different splice variants [For the HD-MY-Z B cell liNFKB2 gene shows an absolute difference of 84.39 TPM between the isoforms ENST00000189444.10 and ENST00000467116.5, the PPP4C  gene shows an absolute difference of 147.32 TPM between the isoforms ENST00000279387.11 and ENST00000561610.1. The HSPD1 (heat shock protein family D (Hsp60) member 1) gene shows an absolute difference in expression of 348.472 TPM between the isoforms ENST00000388968.7 and ENST00000345042.6. PPP4C mediates the repair process of damaged DNA upon double strand break and HSPD1 is involved in B and T cell activation, among other processes. NFKB2 is a member of NF-KB family of proteins and of the TNF signalling pathway. It is involved in transcription, differentiation, apoptosis and inflammatory response. Altogether, these results indicate that a time-dependent change in isoform expression is present in genes with an impact on vital cellular processes. For the L-1236 cell line C, the NFTNFRSF12A and NFKB2 are related to the TNF pathway. Therefore, we investigated the TNF pathway in detail and detected multiple switches in isoform expression over time in several genes. These included TRAF1 and RACK1 (receptor for activated C kinase 1) in the LCL-HO cell line (RACK1 and SHARPIN (SHANK associated RH domain interactor) in the HD-MY-Z cell line (UBB (ubiquitin B) and RPS27A  for the L-1236 cell line  domains are absent. These are critical for folding and stability and are involved in protein-protein interactions. As mentioned above, the cumulative expression of the gene shows little variations (6.93% from 411.96 TPM to 382.52 TPM) whereas the expression of the isoforms shows strong differences within six hours. These findings point to the potential implication of temporal switching events in isoform expression and its impact in the subsequent protein functions.The presence of switches in isoform expression over time and their concentration at specific time points has potential functional implications on the function of the generated protein and the subsequent alterations in the phenotype of the cell. To investigate this aspect in more detail, we performed an analysis at the sequence level with the R package IsoformSwitchAnalyzeR that allows for the integration of data from tools such as CPAT   for codiell line A. For TNthin 9 h B.The ovethe gene . For theThe abundance of time-dependent changes of isoform expression in the TNF pathway  points tTRAF1 in the LCL-HO cell line, TNFRSF12A and RACK1 in the HD-MY-Z cell line and NFKB2 in the L-1236 cell line, which show significant isoform switching behaviour and can regulate the resulting output of the TNF signalling pathway in this cellular model system.Results from our computational analysis revealed that in all of the investigated cell lines, we found a TNF or TNF-related element with temporal changes in isoform production. We identified In the present study, we characterized the existence of time-dependent AS events in HL cell lines  and a non-tumour B lymphoblastoid cell line (LCL-HO) using a computational approach. Stage-specific patterns of AS and splicing factor expression for HL cells from different tumour stages has previously been reported . The autPublished data points to the regulation of AS, and various post-translational processes by the circadian clock pointing to a time-dependency of such molecular mechanisms with implications in cellular function ,27. To iJCHAIN, NME2 and HNRNPH1 genes (for the LCL-HO cell line), CD44, TNFRSF12A and HNRNPA1 (for the HD-MY-Z cell line) and NFKB2, PPP4C and HSPD1 (for the L-1236 cell line), which have an impact in tumorigenesis. Transcript variants of HNRNPH1 were shown to be differentially involved in tumorigenesis. Overall, positive expression of HNRNPH1 is associated with poor tumour differentiation degree [NME2 gene can act as a metastasis suppressor by altering telomere function and telomere length in lung cancer and fibroblasts [CD44 gene [PPP4C gene is required for the correct DNA damage repair functioning upon a double strand break. Moreover, it was shown to be involved in the TNF and NF-kB pathways. It acts as a signalling component connecting the TNF signal to the JNK pathway [HSPD1 encodes a heat-shock family protein chaperone that exhibits elevated expression in Hodgkin’s and large cell lymphoma and is involved in the B and T cell immune response [We further analysed a reduced set of genes that present splicing switches in a time-dependent manner. In particular, out of the top 20 genes with largest expression difference between the isoforms we identified the n degree ,36. The roblasts ,43. CD44D44 gene . ParticuD44 gene . Cancer D44 gene ,46,47. T pathway . HSPD1 eresponse . Altogether, these genes encode for functionally different isoforms involved in vital cellular and tumorigenic processes and hence, the temporal change in their isoform balance could imply subsequent changes in tumour progression. TNFRSF12A and SHARPIN in the HD-MY-Z cell line, NFKB2 in the L-1236 cell line and TRAF1 in the LCL-HO cell line. The TNF receptor superfamily 12A gene (TNFRSF12A) displays a time-dependent isoform generation in the HD-MY-Z cell line. Surprisingly, one of the transcripts for which a temporal isoform generation was detected undergoes NMD, which may influence its functional outcome in time. In cancer, TNFRSF12A can promote angiogenesis and endothelial cell proliferation. It was reported that TNFRSF12A knockout inhibits hepatocellular carcinoma cell proliferation and migration in vitro [TRAF1) showed a temporal pattern of isoform expression. Interestingly, yeast two-hybrid interaction assays revealed that TNFRSF12A binds to members of the TRAF family including TRAF1 and thereby activates the NF-κB signalling pathway [It was recently reported, that splicing isoforms of the main elements within the TNF pathway can have an impact on both inflammation and cancer and, hence, affect the outcome of the pathway . Among tin vitro ,51. TRAFin vitro ,53. Anotin vitro . Anotherin vitro ,56. In t pathway . Compone pathway . Taken together, the results of our study further reinforce the hypothesis, that AS could potentially fine-tune the outcome of the TNF signalling pathway in a time-dependent manner. This adds a hidden layer of temporal regulation in the TNF pathway that will require further experimental studies to fully validate and further evaluate the impact of our findings in tumorigenesis. 2 atmosphere.The Hodgkin lymphoma cell lines, HD-MY-Z (DSMZ—#ACC 346) and L-1236 (DSMZ—#ACC 530), and the B lymphoblastoid cell line, LCL-HO (DSMZ—#ACC 185) were maintained in RPMI 1640 (Gibco) supplemented with 1% penicillin-streptomycin (Gibco) and 10% foetal bovine serum (Gibco) and incubated at 37 °C with 5% CO5 cells/dish and synchronized by fresh medium addition. Cells were collected every three hours, in triplicates and the pellets were stored at −80 °C. Total RNA was isolated using the RNeasy extraction kit (Qiagen), including DNase digestion, according to manufacturer’s instructions. Cells were lysed with 350 µL RLT buffer and the lysate was homogenized. Total RNA concentration was determined using NanoDrop® ND-1000 ultraviolet–visible (UV–Vis) spectrophotometer and stored at −80 °C. A total of 33 time-course data sets (sampling every three hours between ZT12h and ZT42h) was produced for subsequent sequencing.Cells were seeded in 35 mm dishes with a density of 5 × 10For RNA-sequencing the total RNA was processed by Illumina standard protocols to prepare the mRNA-Seq library, followed by deep sequencing in a bi-directional, 75-base mode on an Illumina NextSeq sequencer. The RNA-sequencing was performed in Genomics Core Facility at the European Molecular Biology Laboratory (EMBL) in Heidelberg.p-value cut-off of 0.05 and Bonferroni Hochberg correction for multiple testing against default “universe” background set of genes with a q-value cut-off of 0.2. Disease ontology enrichment was performed with the disease ontology as ontology of choice and p-value cut-off of 0.05 as well as q-value cut-off of 0.5. In all cases, the top 10 enriched terms (if present) were plotted.Enrichment analysis was performed with the DOSE   without a reduction to the most abundant isoforms. For the analysis of coding functionality, we used a coding potential cut-off of 0.725. For the visualization of pairwise comparisons between the STPs we used a difference cut-off of 0.1. Genes with single isoform are removed from the pairwise STP analysis. For the detection of splicing switches over time the TSIS R package (ver. 0.2.0) was used with “mean” as the detection method and a spline degree of 4 to focus on isoforms with a period close to 24 h. Isoform switches were visualized with the TSIS  package d is the average difference of abundances between iiso and jiso in the interval kI, k = 1,2 is defined as: kI| is the number of samples in interval kI and exp is the expression of iiso of sample mIks in interval kI. Computation of abundance differences : MetricThe RNA-sequencing data was submitted to the GEO repository and will be released upon publication of the manuscript.


Overview of current understanding of epigenetic alterations after abiotic stresses during reproductive development in cereals.Abiotic stresses, including heat, drought, cold, flooding, and salinity, negatively impact crop productivity. Various stages during reproductive development are especially sensitive to environmental stresses, which may lead to complete sterility and severe yield losses. Plants exhibit diverse responses to ameliorate stress damage. Changes in DNA methylation, histone modification as well as regulation of small RNA and long noncoding RNA pathways have been shown to represent key modulators in plant stress responses. During reproductive development in cereals, various protein complexes controlling histone and DNA methylation have been identified, revealing conserved and novel mechanisms regulating abiotic stress responses in cereals and other plant species. New findings highlight the role of transposable elements during stress periods. Here, we review our current understanding of epigenetic stress responses during male and female gametophyte formation (germline development), fertilization, early seed devolvement, and seed maturation in cereals. An integrative model of epigenetic responses during reproductive development in cereals is proposed, emphasizing the role of DNA methylation and histone modifications during abiotic stresses. With the human population continuously growing, the demand for food supply is increasing exponentially. Unfortunately, production of cereal grains, including some of the most important crops such as maize, wheat, rice, barley, rye, and oats, is highly vulnerable to abiotic stresses - and long noncoding RNA (lncRNA)-associated regulatory pathways are affected during plant development under stress conditions. These include stages of reproductive development such as gametogenesis and seed development  epigenetic alterations during pre-fertilization processes . Then, we discuss (2) fertilization mechanisms, gamete activation, and early seed development involving pro-embryo and syncytium formation as well as endosperm cellularization. Finally, we cover a stage known as (3) seed maturation, in which starch accumulation, desiccation, and seed dormancy are major processes taking place. Even though reproductive development can be subdivided into several other stages, recent reports have shown the importance of the aforementioned stages or phases as determinant for productivity in cereals , which promotes male germline cell fate and meristem growth  were lacking, suggesting that stress-induced mis-regulation of MSCA1 causes the absence of phasiRNAs in anthers and thus affects a number of unknown target genes . When MAC1 is expressed, a small protein is generated that induces adjacent cells to differentiate into supportive tissues  showed gradual degeneration of epidermal and archesporial cells resulting in anther abortion as a consequence of transcriptional inhibition , which encodes a germline-specific AGO protein that preferentially binds 21-nt phasiRNAs derived generally from intergenic regions. Additionally, MEL1 plays a central role in the progression of meiosis beyond the leptotene stage  from pre-meiosis to meiosis stage is controlled by an increase in di-methylation of lysine 9 of histone 3 (H3K9me2) and a decrease in acetylation of lysine 9 of histone 3 (H3K9) as well as phosphorylation of histone 3 (H3S10) (Liu and Nonomura ZmAGO18b) was pointed out as a possible candidate based on its transcriptional profile . A detailed analysis of QTLs revealed the presence of miRNA targets associated with ABA-responsive genes. Additionally, suppressor of the G2 allele of skp1 (SGT1), a direct target of miRNA166e, was also mapped within the qHT-8 locus  concentration in pollen grains correlates well with pollen sterility and decreased yield  where they germinate and grow toward the ovule to deliver its sperm cell cargo. To which extent this so-called progamic phase of reproduction is affected by abiotic stresses is almost completely unclear. A comparison between pre- and post-pollinated silks in maize identified 30 conserved miRNA families  expression was regulated in a biphasic manner. First, levels of histone H3K4 changed from a di- to a tri-methylation state followed by an increase in acetylation of H3  represents a major regulator of early seed development. During endosperm development in cereals, several active members of PRC2 have been identified  insertion into the 3′-UTR of HSP16.9 affecting its transcription rate  required for maternal allele demethylation and imprinting in the endosperm was reported to be involved in responses to stress. In a drought-tolerant barley variety, increased A recent study performed during rice seed development detected a strong increase in acetylation levels of proteins during seed differentiation and maturation  gene, which is a target of the RdDM pathway (Surdonja et al. Additionally, RdDM pathways have been shown to regulate many developmental processes including seed maturation in cereals (Lanciano and Mirouze Epigenetic mechanisms controlling reproductive development have been shown in the past decade as pivotal regulators of stress responses in cereals. Development and function of female and especially male gametophytes appear to be especially susceptible to abiotic stresses. DNA methylation and small regulatory RNAs including phasiRNAs, lncRNAs, and miRNAs have been shown as repressors of germline initiator genes. Mis-regulation of germline-specific genes during abiotic stresses seems to be a consequence of the lack of the aforementioned epigenetic regulators Fig. .It is still unclear whether the fertilization process itself is affected by stresses and if so, which epigenetic components are the main targets. However, since histone variants are exchanged during zygotic gene activation at non-stress conditions and DNA methylation of imprinted genes is active immediately after fertilization, we speculate that this stage is also affected by abiotic stresses. Proper activity of the PRC2 complex is very critical for seed formation during early stages involved in cell/nuclei divisions, endosperm cellularization, and embryo patterning Fig. . AbioticThese above-described findings now provide exciting prospects since a large number of these processes are crucial for maintenance of yield and reproductive success under abiotic stress conditions. In conclusion, even though plants are exposed to abiotic stresses throughout their life cycle, reproductive development is more vulnerable to abiotic stresses compared to vegetative developmental stages as stress may terminate development and lead to complete sterility. The systematic study of epigenetic regulatory mechanisms under abiotic stress conditions during reproductive development needs to be extended and approached under field conditions, since multiple stresses are frequently coexistent. Inheritable epigenetic changes such as small regulatory mechanisms, histone variants, and DNA methylation are also unexplored in cereals in a trans-generational memory context, since these epigenetic variations might improve stress resilience in the offspring.Finally, although new tissue- and cell-specific methodologies including fluorescence-activated cell sorting, ATAC-seq, and methylome sequencing (e.g., Begcy and Dresselhaus


Leymus chinensis, a perennial rhizomatous grass, and conducted a series of field experiments in situ, including grading sampling of reproductive ramets and different connection forms of vegetative ramets labeled with 15N at four reproductive stages. Reproductive ramet biomass, inflorescence biomass, seed number, seed-setting percentage, reproductive allocation, and reallocation significantly increased with an increase in the number of vegetative ramets connected to tillering nodes, and the plasticity indexes of these six phenotypic characteristics showed similar increasing trends. The amount of nutrients supplied from the connected vegetative ramets to the reproductive ramets was significantly affected by the transfer direction, reproductive stage, and position order of the vegetative ramets. Throughout the sexual reproduction stage, nutrients were preferentially transferred to the acropetal reproductive ramet in L. chinensis populations. The amount of nutrients supplied from the connected vegetative ramets to the reproductive ramets at the milk-ripe stage, when sexual reproduction was most vigorous, was significantly larger than that at other reproductive stages. The amount of nutrients supplied from the spacer vegetative ramet to the acropetal reproductive ramet was significantly larger than that to the basipetal reproductive ramet. The closer the vegetative ramet was to the reproductive ramet, the more nutrients were supplied; the amount of nutrients supplied was significantly negatively related to the position order of the vegetative ramet. We identified the determinant of plasticity in sexual reproduction in clonal plants in a homogeneous environment: physiological integration between ramets within clones. Our results are vital for better understanding the adaptation of populations and even the evolution of species of clonal plants.Phenotypic plasticity is considered a major mechanism that allows plants to adapt to heterogeneous environments. The physiological integration between the interconnected rhizomes or stolons of clonal plants influences the plasticity of such plants in heterogeneous environments. However, the determinants of plasticity of reproductive ramets in clonal plants in homogeneous environments are unclear. Here, we chose  Phenotypic plasticity, or the ability of a given genotype to generate various phenotypes under different environmental conditions , is consClonal plants are widely distributed in numerous ecosystems, such as grasslands and wetlands , and thePrevious studies concerning the effect of physiological integration on the plasticity of clonal plants were mostly carried out in heterogeneous environments. Moreover, what has been confirmed relies on the heterogeneity of the environment . CurrentLeymus chinensis, a typical clonal plant with vigorous belowground rhizomes  to verify that the plasticity in sexual reproduction in L. chinensis clones can be induced by differences in nutrient supply from the connected vegetative ramets in a natural homogeneous environment and b) to reveal the patterns of nutrient supply from the connected vegetative ramets to reproductive ramets in L. chinensis clones. We hypothesize that 1) the phenotypic characteristics and plasticity in these characteristics of reproductive ramets will increase with an increase in the number of vegetative ramets connected to tillering nodes; 2) the amount of nutrients supplied from vegetative ramets with various connection forms to the reproductive ramets will be largest at the vigorous milk-ripe stage; 3) the amount of nutrients supplied from spacer vegetative ramets to acropetal reproductive ramets will be much larger than that to basipetal reproductive ramets; and 4) the closer the vegetative ramet is to the reproductive ramet, the more nutrients will be translocated.In this study, we chose The study was conducted at the Grassland Ecological Research Station of Northeast Normal University in the southern Songnen Plain in Changling County, Jilin Province, Northeast China , in 2018, the fourth year after the experimental plots were set up. This area is characterized by a semi-humid, semi-arid, and temperate continental monsoonal climate with hot and rainy summers and cold and dry winters. The annual mean temperature varies from 4.6 to 6.4°C. The number of days with an average temperature ≥10°C ranges from 120 to 140. The annual accumulated temperature varies from 3,000 to 3,500°C. The annual mean precipitation ranges from 300 to 450 mm, with the majority concentrated in June-September, and the annual evaporation varies from 1,200 to 1,400 mm. The frost-free period is approximately 130–165 days .L. chinensis (Trin.) Tzvel., a perennial rhizomatous C3 grass, is widely distributed in the eastern Eurasian steppe, including northwestern Siberia, western North Korea, the People’s Republic of Mongolia, the Inner Mongolian Plateau, and the Northeast Plain of China . Most often, only one of the ramets interconnected by tillering nodes undergoes sexual reproduction, flowering and seed set, called reproductive ramets; the others only conduct vegetative growth and do not produce flowers, called vegetative ramets. In the Songnen grassland, L. chinensis usually begins turning green in early April, heads in the middle or later period of May, flowers in June, and ripens seeds in mid-July , and adjacent plots were at least 2 m apart. Similarly sized vegetative ramets of L. chinensis  were collected from a natural clone in the study area, and a group of nine ramets was transplanted into each plot with rows 0.5 m apart and ramets 0.5 m apart. All the experimental plots were manually irrigated for several days after transplanting to ensure that the ramets survived. No fertilizer was added to the soil. L. chinensis ramets were not obviously affected by diseases or insect pests throughout the study, and all other plants were regularly pulled by hand from 2015 to 2018. The soil type was sandy loam. The soil of the top 20-cm-thick layer was homogenous, with a total N of 1.01 g kg−1, an organic C of 6.23 g kg−1, a total P of 0.74 g kg−1, a pH of 8.37, and an electrical conductivity of 70.85 μS cm−1.In May of 2015, a total of 30 experimental plots were established. Each plot had an area of 4 mL. chinensis in 2018. Reproductive ramets whose inflorescence top reached approximately 2 cm over the flag leaf sheath and that were interconnected with 1, 2, 3, and ≥4 vegetative ramets by tillering nodes were marked with tags. For each number in the gradient, in each plot, only one reproductive ramet was marked, for a total of 25 reproductive ramets in 25 plots. Five plots were disqualified because the desired number gradient was unavailable. All marked reproductive ramets were harvested together at the dough stage. Reproductive ramet height, floret number, and seed number were measured. Each reproductive ramet was separated into leaves, stem, inflorescence, and seeds. Dry biomass, including all parts of a reproductive ramet, was determined after oven-drying to a constant weight at 65°C.To investigate the effects of the number of vegetative ramets connected to tillering nodes on the phenotypic characteristics of reproductive ramets in a natural homogeneous environment, a marking manipulation was conducted at the early heading stage of in situ-labeling experiment , reproductive stage  was applied, with a urea concentration of 2% g ml−1 and a 15N abundance of 5.18%. The urea solution was daubed gently with a small paintbrush from the base to the tip of the leaf surface to ensure that the total solution was smeared evenly on both sides of the leaf. During brushing, leaves were held with forceps. Labeling was applied once per day over three consecutive days, and meticulous operation with extra care was required to prevent the urea solution from contacting any part of the reproductive ramet or soil. For the control treatment, the 15N-labeled urea solution was replaced by an equal volume of distilled water.Each δ) notion and expressed as follows:At each reproductive stage, the reproductive ramets that were connected to the labeled vegetative ramets were harvested 2 days after labeling. Each ramet was separated into leaf blades, sheath, stem, and inflorescence. The enzyme activity of each part was halted at 105°C for 30 min, and the tissue was dried to a constant weight at 65°C and ground to a fine powder using a ball mill . Approximately 3 mg of sample powder was loaded into a capsule and analyzed using an Elementar Vario EL Cube  coupled to an isotope ratio mass spectrometer , with an overall precision greater than 0.2‰. The isotope value was used the standard delta .where R15N from labeling, the F-ratio was calculated as follows:To calculate the amount of 15N in each sample was then calculated as follows:The amount of translocated Wdry is the dry weight of a sample, B is the nitrogen content per unit mass of a sample, FS is the F-ratio of a sample, and FU is the mean F-ratio of four unlabeled control ramets for each harvest.where P < 0.05.All statistical analyses were performed using SPSS 22.0 software . All data were tested for a normal distribution and homogeneous variances through a Kolmogorov-Smirnov test and Levene’s test, respectively. The significance level was set at One-way ANOVA was conducted to assess the effect of the number of connected vegetative ramets on the phenotypic characteristics of reproductive ramets. The seed-setting percentage was calculated as the percentage of seeds relative to the number of florets, reproductive allocation was calculated as the percentage of inflorescence biomass relative to ramet biomass, and reproductive reallocation was calculated as the percentage of seed biomass relative to ramet biomass. To evaluate the phenotypic plasticity in each characteristic of reproductive ramets interconnected with different number grades of vegetative ramets by tillering nodes, the phenotypic plasticity index (PPI) was calculated as  follows:T is the characteristic mean value in each number gradient.where 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet and to investigate the effects of position order and reproductive stage on the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet. All data on the amount of translocated 15N were log-transformed to meet model assumptions. An independent samples t-test and Duncan’s multiple-range test were used to test for significant differences between means of two and more than two groups, respectively. Estimation of regression curves was used to quantify the relationships between the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet and position order of the acropetal vegetative ramet at the heading stage, flowering stage, milk-ripe stage and full-ripe stage with linear, exponential, power, and logarithmic functions. The model with the lowest Akaike information criterion (AIC) value was selected as the best  or five members (one reproductive ramet and four vegetative ramets connected via one and the same rhizome). In the present study, we detected significant effects of transfer direction on the amount of translocated 15N in the leaf blade, sheath, stem, inflorescence, and whole ramet of the reproductive ramet in L. chinensis with a three-member clonal system  and basipetal translocation  . Most st a clone . Previoue ramets . Howeverl system Table 3.via one and the same rhizome, significant effects of the position order of the connected vegetative ramet on the amount of translocated 15N were found in the leaf blade, sheath, stem, inflorescence, and whole ramet of basipetal reproductive ramets of L. chinensis , National Natural Science Foundation of China (31672471 and 31670427), and the Program for Introducing Talents to Universities (B16011).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.