text
string | predicted_class
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|---|---|---|
We appreciate the reviewer’s comments, and agree that a stable CMG-Mcm10 complex is suggestive that Mcm10 always travels with the replisome. But the available cellular studies that address whether Mcm10 travels with the replisome are not settled in the literature, with reports and data going in both directions. Since we have not shown the persistence of Mcm10 (i.e. koff rate) with CMG at a fork, we discuss the possibility that CMG might travel with the fork all the time in the Discussion, as requested, but we also note that we cannot rigorously make that conclusion from the data of the current report.
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other
| 99.6 |
4) Figure 1C cartoon is a bit misleading. The MCM should be depicted as a 3 tier ring: from the bottom, ATPase, A-C tier and smaller B tier. Duplex DNA is engaged by B-tier only. At the moment it appears that fork nexus is found at the ATPase/A-C tier interface, which is not the case.
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other
| 99.9 |
We thank the reviewers for pointing this out. We did not intend to draw the unwinding point as far down as we did, and this was an artistic flaw of the original figure. We have now redrawn the figure to show a much smaller amount of dsDNA entering the CMG. We did not redraw the cartoon to have 3 tiers because describing the A, B, C domains of the N-tier is beyond the scope of the Introduction. We have dealt with these aspects in the structural study and in a few other reviews that are submitted and underway.
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other
| 99.9 |
5) Figure 2A, it would be good to include a control where Mcm10 is incubated with a fork in the absence of CMG. Also, one caveat of this experiment is that Mcm10 is a single-stranded DNA binding protein and the stimulation of the DNA unwinding activity might simply be due to single-stranded DNA trapping (effectively the same role of a DNA capture strand). It would be good to test whether another single-stranded DNA binding protein (e.g. SSB) has a similar effect on DNA unwinding by the CMG. Also, what is the supershifted band visible e.g. in lanes 5, 8 and 11? It would be good to acknowledge this band, since it is present in many unwinding gels throughout the paper.
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other
| 88.75 |
– We long ago performed the experiments that ask whether RPA or SSB might stimulate CMG unwinding prior to submitting the original manuscript, and have now included this data as a Figure 2—figure supplement 2. We should note that SSB/RPA can’t be added to reactions before CMG as it prevents CMG loading (as we published earlier). Thus, we let the CMG helicase reaction proceed for 10 min, and then add either Mcm10, RPA or SSB. The result shows that Mcm10 provides strong stimulation of the CMG helicase while neither RPA nor SSB affect CMG activity.
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| 99.9 |
“Control reactions with Mcm10 alone, lacking CMG, show no unwinding activity (Figure 2—figure supplement 1). Reactions with Mcm10 sometimes give a supershift of some of the substrate (e.g. lanes 5, 8, 11, 12 in Figure 2A), which we interpret as a gel shift of Mcm10 that remains bound to DNA, as Mcm10 is a known DNA binding protein (Du et al., 2012; Robertson et al., 2008; Warren et al., 2008). To determine if other DNA binding proteins such as RPA or E. coli SSB stimulate CMG, we performed experiments in which CMG unwinding was initiated and then either Mcm10, RPA or SSB was added. The results show that the stimulation of CMG is specific to Mcm10 and that RPA and SSB do not substitute for Mcm10 (Figure 2—figure supplement 2).”
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| 100.0 |
6) Figure 2D. GINS subunits are notoriously difficult to stain. It is a pity that in the CMG-Mcm10 gel appears to be cut so that the GINS subunits are not visible. From looking at this gel alone one cannot rule out the possibility that Mcm10 and GINS compete for the same binding site on the MCM. In Figure 2—figure supplement 1 the MonoQ profile is shown where Sld5 is detected, however not Psf1-3. It is remarkable that the CMG-Mcm10 complex is stable at 300mM KCl concentration (indeed convincingly shown in the high salt FLAG pulldown experiment), however the Q column experiment alone does not rule out the possibility that Sld5 and MCM-Cdc45-Mcm10 simply elute at the same salt concentration from an ion exchange column. Could the authors produce a gel where all of the CMG components are seen together with Mcm10, after FLAG elution?
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| 99.94 |
We appreciate the reviewer’s concerns and certainly agree that the GINS are notoriously difficult to stain. We have rerun the gels, but we should also point out that while this project was being performed, Mcm10 was demonstrated in the Bell lab to preferentially bind CMG, containing GINS, Cdc45 and Mcms (Lõoke, et al., 2017). However, we have re-run FLAG and MonoQ experiments and re-run gels to the best we could, and have added those gels and the following text to the manuscript. We also note that Steve Bell’s recent paper on Mcm10 (cited herein) demonstrates that Mcm10 stabilizes the CMG complex.
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| 98.94 |
“To determine the stoichiometry of GINS in the CMG-Mcm10 complex, a Flag purification of CMG-Mcm10 was analyzed in a 10% PAGE gel followed by densitometric analysis. The result indicates the following stoichiometry: 1.0 Mcm2-7, 1.2 Mcm10, 0.87 Sld5, and 2.6 Psf1+Psf2 (they co-migrate for an average of 1.3 each). The Psf3 runs at the dye front and could not be quantitated. FLAG columns capture some free Cdc45 and thus overestimate the Cdc45-FLAG subunit; MonoQ analysis removes free Cdc45 (see Figure 2—figure supplement 4).”
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“The GINS subunits of the CMG-Mcm10 complex ran off the gel, but a previous study demonstrated that Mcm10 preferentially associates with the entire CMG complex (Lõoke et al., 2017). This is supported by densitometry analysis of gels showing GINS subunits in both FLAG and MonoQ purified CMG-Mcm10 complexes (Figure 2—figure supplements 3 and 4).”
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| 100.0 |
We note to reviewers (not in the text), that scans of Commassie stained bands are only approximations because different proteins can take up different amounts of stain, but that the results overall indicate that Mcm10 binds CMG without displacing the GINS and Cdc45 accessory factors.
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| 99.8 |
We have also recently performed cross-linking/mass spectrometry on CMG-Mcm10 complex, and find that Mcm10 runs across the surface of two of the GINS subunits and the Cdc45 subunit, and wraps around 3 of the six Mcm2-7 subunits. The CX-MS results are not included here, because they are part of our study on the structure of CMG-Mcm10, but they confirm that GINS and Mcm10 are associated with CMG, and that Mcm10 binds a lot of the CMG subunits, which probably accounts for Steve Bell’s observation in his recent study that Mcm10 preferentially binds the complete CMG, and facilitates formation of the CMG complex (Lõoke, et al., 2017).
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7) Figure 3. The observation that CMG plus Mcm10 is much better at unwinding longer stretches of duplex DNA compared to CMG alone is interesting. Why do the authors think that a fork with 160bp duplex is a better substrate than the 50bp duplex for CMG-Mcm10?
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| 98.6 |
We apologize for the misconception in our text. We did not mean to imply that the fork with a 160bp duplex is a better substrate than a fork with a 50bp duplex. We meant to say that these appear to be equal substrates for CMG-Mcm10. We have clarified this point in the text of the revised manuscript. We have added the following statement to the Results:
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| 99.9 |
8) Figure 5—figure supplement 1 lanes 11-13. CMG-Mcm10 can clearly bypass a leading strand roadblock, however the authors do not comment on this remarkable observation. Is SA kicked off of the DNA or is there a more complicated leading strand roadblock bypass mechanism to be uncovered? Using DNA-Protein crosslink (methyltransferase?) as a roadblock would address this issue. If SA is kicked off of the leading strand template, this might simply mean that the bypass effect observed here is due to a stimulation of the ATPase activity (CMG-Mcm10 powers through the roadblock). Could the authors address this point? Does Mcm10 stimulate ATP hydrolysis by MCM to a different extent compared to e.g. MTC, which the authors already use as an informative control in this study?
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| 99.5 |
From these experiments it is not clear that streptavidin remains bound to the substrate after CMG passage, as invoked by the bypass mechanism proposed by the authors. This can be addressed by placing the 32P-label on the modified strand of the DNA substrate to observe the generation of single-stranded streptavidin-DNA products, and by performing the reactions in the presence of excess free biotin to trap displaced streptavidin. This seems particularly relevant, as the experiment in Figure 5—figure supplement 1 detects significant Mcm10-dependent bypass of blocks even on the leading strand – how do the authors explain this latter observation?
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| 99.94 |
We appreciate this comment, and in fact did the excess biotin trap experiment before submission for the lagging strand streptavidin blocks. On hindsight we should have included that data in the original submission. In the revised manuscript, we perform both leading and lagging experiments using a biotin trap (and 32P-label on the biotinylated strand), and using the forked DNAs with equal 6bp spacing between the two biotin-streptavidins for both leading and lagging strands. The results show that Mcm10 enables bypass of the lagging strand blocks without displacing the streptavidin blocks, and that the leading strand streptavidins are in fact displaced from DNA (there was less efficient leading strand read through with the 6 bp spacing compared to the earlier 14 bp spacing). These data are now presented in a newly added Figure 6 and supplement to Figure 6, which shows the effectiveness of the biotin trap. The modified manuscript reads as follows:
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| 98.75 |
“The GINS subunits of the CMG-Mcm10 complex ran off the gel, but a previous study demonstrated that Mcm10 preferentially associates with the entire CMG complex (Lõoke, et al., 2017). This is supported by densitometry analysis of gels showing GINS subunits in both the FLAG and MonoQ purified CMG-Mcm10 complexes (Figure 2—figure supplements 3 and 4).”
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| 100.0 |
Medicinal plants have long been used to treat various diseases including cancers for thousands of years. In contrast to the conventional cancer chemotherapy agents targeting single molecule, the mixture of phytochemicals is able to target multiple-molecules involved in the same pathway or several pathways responsible for cancer development, and exerts better therapeutic efficacy and lower side effects1. Dried 4- to 5-year-old roots of Saussurea lappa, known as Mu-xiang, are commonly used as medicine to treat breast cancer and breast hyperplasia in China, Japan and India2. Our previous study demonstrated that volatile oil from Saussurea lappa root (VOSL), sesquiterpene lactones-rich fraction, is responsible for the anti-breast cancer activity of Mu-xiang3. Gas chromatography-mass spectrometer (GC-MS) and liquid chromatography-mass spectrometer (LC-MS) analyses revealed that Costunolide (Cos) and Dehydrocostuslactone (Dehy), two natural sesquiterpene lactones, are the main ingredients of VOSL. Moreover, the combination treatment of Cos and Dehy (CD) showed synergistic anti-breast cancer efficiency both in vitro and in vivo34.
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study
| 99.94 |
Much evidence indicates that the α,β-unsaturated carbonyl group in the α-methylene-γ-butyrolactone (Fig. 1) moiety of Cos and Dehy may play crucial roles through conjugation with SH-groups of target proteins to exert various biological activities, such as anti-inflammatory, anti-cancer, anti-virus, anti-oxidant, anti-diabetes, anti-ulcer, and anthelmintic activities, etc.5, of which, the anti-cancer activities and associated molecular mechanisms of Cos or Dehy have been reported in recent years, including inhibiting cancer cell proliferation6, accelerating apoptosis7, inducing cancer cell differentiation8, inhibiting metastasis and invasion9, reversing multidrug resistance10, restraining angiogenesis11. Our previous studies had demonstrated that VOSL has better anti-breast cancer efficacy and lower side effects than Cos or Dehy in vivo3, however, to the best of our knowledge, the synergistic anti-cancer molecular mechanism of Cos and Dehy (CD) in VOSL has not yet been studied.
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| 99.94 |
Protein phosphorylation is a reversible protein post-translational modification which likes a molecular switch controlling important biological processes such as cell division, growth, differentiation, and death. Its misregulation is often associated with many human diseases, including cancer12. The research results from Choi et al. showed that sesquiterpene lactones can act as phosphatase inhibitors13. Therefore, we speculated that the cytotoxicity of VOSL or CD towards human breast cancer cells should be associated with protein phosphorylation pathways. Developments of phosphopeptide enrichment technologies along with improvements in mass spectrometer sensitivity, protein database and bioinformatics algorithms, have facilitated the qualitative and quantitative analyses of phosphopeptides from complex cell extracts and greatly revolutionized the fields of cell biology and cell signaling14. Currently, TiO2 has been considered as the most effective enrichment material for phosphopeptides15, and isobaric tags for relative and absolute quantification (iTRAQ) technology has been widely used to proteome research. In this study, we explored the anti-breast cancer molecular mechanism of VOSL and CD through TiO2-based enrichment of phosphopeptides and iTRAQ-based liquid chromatography and tandem mass spectrometry (LC-MS/MS) proteomics, coupled with Western blot validation.
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| 99.94 |
Two sets isotope-labelled mixed samples (set one is Ctr (114) and Cos (117); set two is Ctr (114), Dehy (115), CD (116) and VOSL (117)) were analyzed by Nano LC–Q/TOF MSE tandem mass spectrometry and identified a total of 430 proteins in set one (Supplementary Table S1), and 469 proteins in set two (Supplementary Table S2). Only protein quantification data with relative expression of >1.5 or <0.66 were chosen as differentially expressed proteins (Supplementary Table S3). The numbers of differentially expressed proteins in the Cos, Dehy, CD, and VOSL-treated group were 67, 59, 38, and 47, respectively. The differentially expressed proteins were imported into the IPA software for function annotation and interaction network analyses. The interaction networks of differentially expressed proteins in the Cos-treated group enriched 27 proteins (Supplementary Fig. S1A), thereinto, 15 proteins, which were APEX1, C1QBP, COL1A1, FAM162A, FXR1, HSPB8, HSPD1, LMNA, MAPT, NPM1, PGRMC1, RPS3, SET, SFN, STMN1, involved in the physiologic functions of cell death and survival. The interaction networks of differentially expressed proteins in the Dehy-treated group enriched 26 proteins (Supplementary Fig. S1B), of which there were 14 proteins, API5, BID, CFL1, EZR, FASN, GNB2L1, HMGB1, HSPB1, MAPT, SFN, SMARCB1, SON, TOP1 and YWHAZ, involved in the physiologic functions of cell death and survival. The interaction networks of differentially expressed proteins in the CD-treated group enriched 22 proteins (Supplementary Fig. S1C), among which there were 4 proteins, MAPT, CFL1, FLNB and SMARCA4, involved in the physiologic functions of cellular assembly, organization and cell cycle. The interaction networks in the VOSL-treated group enriched 23 differentially expressed proteins (Supplementary Fig. S1D), in which there were 11 proteins, API5, BID, GNB2L1, HSPB8, PARP1, RBM25, SFN, SND1, SON, TXN and UBR4, involved in the physiologic functions of cell death and survival.
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| 100.0 |
As we described above, Cos and Dehy are both natural sesquiterpene lactones, and they account for nearly 75% of VOSL by weight. Therefore, there should be some common differentially expressed proteins among the Cos, Dehy, CD and VOSL treated groups. Our results validated this speculation, 14 common up-regulated proteins, 20 common down-regulated proteins, and 43 common differentially expressed proteins were observed (Fig. 2A–C) and their alterations were depicted as a heatmap in Fig. 2D (use fold change, relative to Ctr). The results of cluster analysis revealed that the VOLS-treated group shared the most differentially expressed proteins with the CD-treated group, which further demonstrated that CD are the most important anti-breast cancer ingredients in VOSL and share the same pharmacological mechanisms with VOSL.
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| 100.0 |
The differentially expressed proteins at different treatment groups were imported into the IPA software for pathway analysis, the results demonstrated that the top pathway is c-Myc mediated apoptosis signaling and 14-3-3-mediated signaling for Dehy or Cos treatment, respectively (Fig. 3A and B), VOSL and CD shared the common top pathways, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling (Fig. 3C).
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| 100.0 |
Pathway analysis based on the IPA software demonstrated that Cos, Dehy, CD or VOSL treatment affected some important signaling pathways in breast cancer cells, such as c-Myc mediated apoptosis signaling, 14-3-3-mediated signaling, and PKA signaling, therefore, Western blot analysis was used to validate these results. The results revealed that Cos, Dehy, CD and VOSL treatment all did not significantly regulated the expression of AKAP8 (Fig. 4A and B), however, CD and VOSL treatment can up-regulate the expression of p53 and down-regulate the expression of c-Myc significantly. The ratios of p53/c-Myc were increased dose-dependently in the four test groups compared with the control group, and the ratios of p53/c-Myc in the CD and VOSL treated groups were obviously bigger than those in the Cos or Dehy treated groups (Fig. 4C and D). p53 is a well-known tumor suppressor protein, its overexpression can induce up-regulation of Bax and mitochondria-dependent apopotosis16. In present study, we also found that the ratio of BAX/BCL-2 was increased (Fig. 4E and F) and the ratio of p-BID/BID (Fig. 4G and H) was decreased dose-dependently in the four test groups compared with the control group, which meant that Cos, Dehy, CD and VOSL all can induce MCF-7 and MDA-MB-231 cell apoptosis by the mitochondria-dependent intrinsic pathway.
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study
| 100.0 |
Numerous studies showed that activation of AKT is positively correlated with cancer development, and c-Jun NH2-terminal kinase (JNK) can antagonize AKT-mediated survival signals by phosphorylating 14-3-3. In this study, the phosphorylation level of AKT was down-regulated (Fig. 4I and J) and the phosphorylation level of 14-3-3 was up-regulated (Fig. 4K and L) dose-dependently, with no obvious changes of the total AKT and 14-3-3 levels in the four test groups compared with the control group, and the ratios of p-AKT/AKT in the CD and VOSL treated groups were obviously lower than that in the Cos and Dehy treated groups, and the ratios of p-14-3-3/14-3-3 in the CD and VOSL treated groups were obviously higher than that in the Cos and Dehy treated groups.
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| 100.0 |
Adenyl cyclases (ACs) can convert adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), which is necessary for the activation of PKA. The IPA analysis revealed that the signal pathway Gβγ/AC2/4/cAMP/PKA was inhibited by VOSL or CD. AC2 is distributed in brain and lung tissues, however, AC4 is widely distributed in various tissues. Therefore, we determined the levels of AC4 (Fig. 5A–C) and its catalysate cAMP (Fig. 5D–F). The results showed that the levels of AC4 and cAMP were decreased dose-dependently in the four test groups compared with the control group, and the levels of AC4 and cAMP in the CD and VOSL treated groups were obviously lower than those in the Cos and Dehy treated groups.
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| 100.0 |
Western blot analysis demonstrated that Cos, Dehy, CD, and VOSL treatment all can increase the phosphorylation level of 14-3-3 protein in breast cancer cells. As 14-3-3 protein is a G2/M checkpoint regulator, which can regulate cell cycle progression and promote cell apoptosis171819. Therefore, we thought Cos, Dehy, CD, and VOSL treatment should induce breast cancer cell cycle arrest. The results of cell cycle analysis were shown in Fig. 6A and B, which verified our speculation. IC10 of tested compound treatment did not change the progression of breast cancer cell cycle, interestingly, IC30 of Cos or Dehy treatment was apt to induce G2/M phase arrest, however IC50 of Cos or Dehy treatment was apt to induce S phase arrest. Moreover, IC50 of CD can significantly induce S phase arrest for MCF-7 cells and significantly induce S phase and G2/M phase arrest for MDA-MB-231 cells. IC50 of VOSL treatment can significantly induce S phase and G2/M phase arrest for the two breast cancer cell lines.
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| 100.0 |
IPA analysis results revealed that apoptosis induction for MCF-7 cells is an important anti-cancer molecular mechanism of Cos, Dehy, CD, and VOSL. In this study, we further validated the IPA analysis results based on Annexin V-FITC/PI apoptosis analysis. The results of cell apoptosis analysis were shown in Fig. 6C and D. Cos, Dehy, CD and VOSL treatment all can dose-dependently induce MCF-7 cell and MDA-MB-231 cell apoptosis, moreover, the effects of apoptosis induction in the CD or VOSL-treated group are stronger than those in the Cos or Dehy-treated group.
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| 100.0 |
Our previous study demonstrated that VOSL and its main active ingredients can suppress the growth of estrogen receptor positive breast cancer MCF-7 xenografts3. In this study, we used the estrogen receptor negative breast cancer MDA-MB-231 xenograft model to evaluate further the anti-breast cancer efficiency of VOSL in vivo. The results revealed that VOSL and its main active ingredients also can suppress the growth of MDA-MB-231 xenografts, and VOSL and CD exhibited better anti-breast cancer activity than Cos or Dehy treatment alone (Fig. 7A and B). The inhibitory rates of VOSL, CD, Dehy, and Cos on MDA-MB-231 xenografts are 62.75%, 54.94%, 31.63%,and 27.85%, respectively, after intraperitoneal injections for 30 times. In addition, the expression of several key molecules, such as p53, c-Myc, p-AKT, p-14-3-3, in tumor tissues was determined by immunohistochemistry. The results demonstrated that the expression levels of c-Myc and p-AKT were all reduced and the expression levels of p53 and p-14-3-3 were all elevated in the treatment groups compared with the negative control group. Moreover, the CD or VOSL treated groups showed more obvious expression differences of these molecules than the Cos or Dehy treated groups (Fig. 7C and D). The results were consistent with the in vitro results. Therefore, we concluded that combination treatment of Cos and Dehy inhibits breast cancer through c-Myc/p53 and AKT/14-3-3 pathway.
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| 100.0 |
Our previous researches revealed that Cos and Dehy in VOSL exhibited synergistic anti-breast cancer efficiency both in vitro and in vivo. In this study, we tried to investigate their molecular mechanisms. Increased proliferation capacities, uncontrolled cell cycle progression and apoptosis inhibition are the hallmark of cancer. Accordingly, the agents targeting one or more of these processes should be ideal cancer chemopreventive candidates5. Our research results demonstrated that VOSL, sesquiterpene lactones-rich fraction, can inhibit MCF-7 cell proliferation, induce cell cycle arrest and promote apoptosis through c-Myc/p53 signaling pathway and AKT/14-3-3 signaling pathway.
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| 100.0 |
Much evidence showed that c-Myc plays a critical role in the control of cell proliferation, regulation of cell cycle, and serves as a link between proliferation and cell death by inducing p53-dependent apoptosis2021. c-Myc has been documented to be both a positive and a negative signal for induction of apoptosis22. It is well known that overexpression of c-Myc induces normal cell apoptosis23. However, down-regulation of c-Myc expression may be mandatory for induction of apoptosis in many cancer cells, such as leukemia cells24, prostate cancer cells25, lung cancer cells26, and liver cancer cells27. c-Myc is frequently overexpressed in cancer cells28, enhanced expression of c-Myc will lead to activation of Cdk/Rb/E2F pathway, which is critical for cell cycle progression from G1 into S phase20. Moreover, c-Myc plays an important role in controlling various genes endcoding protein-synthesis components and regulating the expression of critical proteins in DNA replication machinery2930. Therefore, its overexpression can activate the general apparatus for cellular metabolism and promote the process of DNA replication, so as to prepare cancer cell for continued proliferation. Conversely, deregulated c-Myc expression results in S phase arrest and cell apoptosis20. In addition, down-regulation of c-Myc expression can significantly decrease telomerase activity and inhibit growth of cancer cells3132. Therefore, reduction of c-Myc expression has been considered as a potential therapeutic strategy for cancer33. In the present study, Cos, Dehy, CD and VOSL treatment all can reduce the expression of c-Myc, inhibit MCF-7 cell proliferation and induce its apoptosis.
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| 99.94 |
The intrinsically dual nature of c-Myc function in growth and apoptosis and c-Myc-mediated apoptosis in normal cells requires wild-type p5334, however, the mechanisms of c-Myc-induciable apoptosis and how c-Myc and p53 involved in cancer cell apoptosis are not fully clarified. p53 is a well-known tumor suppressor protein, its overexpression can induce up-regulation of BAX and mitochondria-dependent apoptosis35. Our results were consistent with the references. Dehy, CD and VOSL treatment all can up-regulate the expression of p53, and increase the ratio of BAX to BCL-2. BAX and BCL-2 are both the BCL-2 family members, which serve as critical regulators of the mitochondrial-dependent apoptotic pathway. Thereinto, BCL-2 negatively regulates apoptosis and promotes cell survival, whereas BAX acts as a positive regulator of apoptosis to stimulate mitochondrial damage. The rise in ratio of BAX to BCL-2 will cause an opening of the mitochondrial permeability transition pore, which results in releasing pro-apoptotic proteins from the intermembrane space into the cytosol and triggering the mitochondrial-dependent apoptotic pathway3637. Moreover, phosphorylation of 14-3-3 was dramatically increased and phosphorylation of BID was decreased in the VOSL-treated group. Phosphorylation of 14-3-3 will induce dephosphorylation of BAD. Dephosphorylated BAD and dephosphorylated BID were translocated to mitochondria, where they associate with Bcl-2/Bcl-x(L) to induce the mitochondrial-dependent apoptosis38. Taken together, CD and VOSL treatment can up-regulate the expression of p53, down-regulate the phosphorylation levels of BID and BAD and increase the ratio of BAX to BCL-2, to trigger the mitochondrial-dependent apoptotic pathway.
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| 100.0 |
14-3-3 proteins are a family of evolutionary conserved modulator proteins, which regulate multiple signaling pathways involved in mitogenesis, cell cycle progression, and apoptosis in cells through binding to specific Ser/Thr-phosphorylated motifs on target proteins39. It has been considered as an integration point which integrates a variety of apoptotic and survival signals to adjudicate cell survival or death38. Mammals express seven 14-3-3 isoforms which can form homo and hetero dimers. Upon target binding, 14-3-3 proteins can affect the function of target protein by modulating the enzymatic activity of target protein, its protein stability, cellular localization or its association with other proteins. AKT is a central mediator of the PI3K/AKT pathway, its activation was positively correlated with cancer development404142. Accumulating evidence showed that many AKT targets are also regulated by 14-3-3, including BAD43, TSC244, p27Kip145, YAP46, GSK347, PRAS4048, and LKB149. This sharing of targets is due to the overlap between the recognition motifs of AKT and 14-3-3: RxRxxS/T for AKT and RSxpS/TxP for 14-3-340.
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| 99.94 |
In our study, three isoforms of 14-3-3 proteins, 14-3-3σ (SFN), 14-3-3β (YWHAB) and 14-3-3ζ (YWHAZ), were enriched in 14-3-3-mediated signaling pathway by IPA analysis, and Western blot analysis demonstrated that the phosphorylation of 14-3-3 in MCF-7 cells and MDA-MB-231 cells was obviously increased after Cos, Dehy, CD or VOSL treatment. c-Jun NH2-terminal kinase (JNK) can antagonize AKT-mediated survival signals by phosphorylating 14-3-3. Research results from Choi et al. revealed that Cos treatment can activate JNK and induce apoptosis in Human Leukemia Cells50. Therefore, we proposed that Cos, Dehy, CD and VOSL treatment all can activate JNK and inactive AKT in breast cancer cells, and then the activated JNK will promote the phosphorylation of 14-3-3, which resulted in releasing the proapoptotic proteins, such as BAD and FOXO, and enhancing the activity of tumor suppressor, such as LKB1, to antagonize AKT-mediated survival signals, and finally to induce cancer cell apoptosis.
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| 100.0 |
Protein kinase A (PKA) is a holoenzyme, which composes of two regulatory subunits (R) and two catalytic subunits (C). The two C-subunits are maintained in an inactive conformation by an R-subunit dimmer51. Elevated intracellular cAMP binds to the R-subunit of PKA causing phosphorylation of C-subunit of PKA, and then the activated C-subunit of PKA phosphorylates a range of substrate proteins on serine/threonine residues to govern many biological processes in cells52. The broad-substrate specificity of PKA is directed toward specific intracellular substrates by a multigene family of A-kinase anchor proteins (AKAPs), which target PKA to distinct subcellular loci and coordinate multiple signaling enzymes in supramolecular complexes53. In the present study, IPA analysis revealed that VOSL or CD treatment inhibits the PKA signaling pathway. The flux of cAMP is governed by two sets of enzymes: adenyl cyclase (AC) and phosphodiesterase (PDE), the former is activated by G-proteins to synthesize cAMP from ATP, and the later terminates cAMP signaling by hydrolyzing it to AMP54. Moreover, accumulating documents demonstrated that in addition to anchoring PKA many AKAPs contribute to the local degradation of cAMP by co-localizing PDEs55. Therefore, we proposed that CD or VOSL can act as a G protein-coupled receptor (GPCR) inhibitor to inhibit G-protein activity and decrease cAMP synthesis, and the decreased levels of AC4 and intracellular cAMP in the CD- and VOSL-treated groups supported this deduction. Meanwhile, CD or VOSL treatment might up-regulate the phosphorylation level of AKAP8 with no obvious changes of the total AKAP8, and increase the local degradation of cAMP by co-localizing PDE4A5657. The decreased intracellular cAMP would cause inactivation of PKA and its downstream proteins, such as PDE, BAD, HSL, PHK, TH, NAFT, and Filamin, in turn affect the metabolic energy, lipolysis, glycolysis, tyrosine metabolism, and cytoskeletol regulation in cancer cells (Fig. 3C). These results are consistent with our previous report that is VOSL or CD treatment was able to attenuate the metabolic perturbation in energy metabolism, lipid metabolism, glycolysis, and tyrosine metabolism of MCF-7 xenograft mice3.
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study
| 100.0 |
Taken together, VOSL contained multiple anti-cancer ingredients, at least Cos and Dehy, which targeted multiple signaling pathways, at least c-Myc/p53, AKT/14-3-3 and PKA signaling pathways to exhibit synergistic anti-breast cancer efficiency, and our previous study demonstrated that VOSL and CD shows better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo3. Therefore, it is expected that VOSL and CD may serve as novel anti-tumor agents in prevention and treatment of breast cancer.
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study
| 99.94 |
The annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit was from MultiSciences Biotech (Shanghai, China). 4-Plex iTRAQ reagent was obtained from Applied Biosystems (Framingham, MA). Radio-Immunoprecipitation Assay (RIPA) lysis buffer, phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktails and phosphatase inhibitor cocktails were purchased from Boster Biotech (Wuhan, China). Bradford Protein Assay Kit was purchased from Beyotime Biotech (Shanghai, China). Costunolide (Cos) and Dehydrocostus lactone (Dehy) (>98.0% purity) were purchased from Shanghai Yuanye Biotech (Shanghai, China). Nonphosphorylated peptides LY-6 (H-Leu-Thr-Arg-Pro-Arg-Tyr-OH), DE-11 (H-Asp-Ala-Glu- Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-OH), and phosphorylated peptides LY-6p (H-Leu-Thr-Arg -Pro-Arg-{pTyr}-OH), DE-11p (H-Asp-Ala-Glu-Phe-Arg-His-Asp-{pSer}-Gly-Tyr-Glu- OH) (>95.0% purity) were obtained from GL Biochem (Shanghai, China). cAMP (>98.0% purity) and other reagents used were purchased from Sigma-Aldrich (WI, USA).
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other
| 99.9 |
Rabbit anti-human phospho-AKT (Thr308), rabbit anti-human total-AKT, rabbit anti-human p53, rabbit anti-human c-Myc, horseradish peroxidase-conjugated sheep anti-rabbit IgG antibodies were from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA). Mouse anti-human glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was purchased from Bio-tech (Kangchen Bio-tech, Shanghai, China). Rabbit anti-human 14-3-3, rabbit anti-human phospho-14-3-3 β + ζ (Ser184 + Ser186), rabbit anti-human AKAP8, rabbit anti-human total-BID, rabbit anti-human phospho-BID (Ser61), rabbit anti-human BAX, rabbit anti-human BCL-2, rabbit anti-human adenyl cyclase 4 (AC4) were purchased from Abcam (Cambridge, UK).
|
other
| 99.9 |
An illustration of the experimental workflow as well as the data analysis for this study is shown in Fig. 8. The proteins from five experimental groups, including the control group (Ctr), Cos treated group, Dehy treated group, CD (Cos/Dehy = 1/2, w/w; simulating the composition ratio of VOSL) treated group, and VOSL treated group, were harvested and quantified. The quantified proteins were reduced and alkylated, and then digested into peptides. After TiO2-based enrichment, the phosphopeptides in each group were labeled by 4-plex iTRAQ reagent separately. The labeled phosphopeptides were mixed into two pools, thereinto, one pool was consisted of Cos-treated group (labeled with m/z 117 isotope ion) and Ctr group (labeled with m/z 114 isotope ion), and the other pool was consisted of Ctr group (labeled with m/z 114 isotope ion), Dehy-treated group (labeled with m/z 115 isotope ion), CD treated group (labeled with m/z 116 isotope ion), and VOSL treated group (labeled with m/z 117 isotope ion). The resulting phosphopeptide pools were desalted and then injected into liquid chromatography- tandem mass spectrometry (LC-MS/MS) system. The phosphopeptides in each group were relatively quantified by reporter ions and identified based on sequence information from MS/MS. Identified differential expression proteins were further analyzed using Ingenuity Pathways Analysis (IPA) (version 9.0) (Ingenuity® Systems, http://www.ingenuity.com) to statistically determine the functions and pathways most strongly associated with the protein list. Finally, the results of bioinformatics analysis were validated by cell cycle and apoptosis experiments, and Western blot experiments.
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study
| 100.0 |
The extract of Saussurea lappa root was prepared as previously described4. Briefly, 10 g of Saussurea lappa roots were crushed into powder, and then was extracted with 100 mL hexane by sonication. The filtrates were evaporated to get VOSL. Analytic results from comprehensive two-dimensional gas chromatography- time-of-flight mass spectrometry (LECO Corporation, St Joseph, MI, USA) indicated that Cos and Dehy are the main ingredients of VOSL, accounting for nearly 72% of VOSL by weight (Supplementary Fig. S2 and Table S4). Commercial pure Cos, Dehy, or test sample of VOSL were dissolved in dimethyl sulfoxide (DMSO) to 10 mg/mL as a stock solution. According to the previous results of MCF-7 cell proliferation assays, 10%, 30% and 50% maximal inhibitory concentrations (IC10, IC30 and IC50) of Cos were calculated to be 0.9, 1.3 and 2.2 μg/mL, respectively. IC10, IC30 and IC50 of Dehy were 0.7, 1.1 and 1.7 μg/mL, respectively. IC10, IC30 and IC50 of CD were 0.4, 0.9 and 1.4 μg/mL, respectively. IC10, IC30 and IC50 of VOSL were 1.5, 2.4 and 3.3 μg/mL, respectively4. Moreover, according to the previous results of MDA-MB-231 cell proliferation assays, 10%, 30% and 50% maximal inhibitory concentrations (IC10, IC30 and IC50) of Cos were calculated to be 1.1, 2.3 and 4.2 μg/mL, respectively. IC10, IC30 and IC50 of Dehy were 0.8, 1.5 and 3.3 μg/mL, respectively. IC10, IC30 and IC50 of CD were 0.7, 1.3 and 2.8 μg/mL, respectively. IC10, IC30 and IC50 of VOSL were 1.3, 2.8 and 4.6 μg/mL, respectively.
|
study
| 100.0 |
Human breast cancer MCF-7 and MDA-MB-231 cells were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China) on November 2015, which were authenticated and tested by the short tandem repeat (STR) method. Cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% fatal bovine serum (FBS) (Hyclone, Thermo Scientific, UT, USA). Exponentially growing cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C.
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study
| 99.94 |
MCF-7 cells were trypsinized and seeded into cell culture dishes (15 cm in diameter) at a density of 3 × 106 cells per dish. The cells were cultured for 12 h to allow their adhesion to the dish, and then replaced with fresh medium containing IC50 of Cos, Dehy, CD, or VOSL, respectively. Whereas, the same volume of fresh medium without the test compounds was added to the cells as blank control. All culture dishes were then incubated for 48 h. Biological replicates for each group were performed in duplicate.
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study
| 100.0 |
Cells were cultured for 48 h, the culture medium was aspirated, and the cells were rinsed 3 times with ice-cold phosphate buffer solution (PBS). The cells were harvested and softly homogenized in an ice-cold RIPA Lysis Reagent (BOSTER, Wuhan, China) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (BOSTER, Wuhan, China), sonicated for 400 W × 4 min (6 s on, 4 s off), and centrifuged at 20,000 × g for 15 min. The supernatant containing the total MCF-7 cell proteins was precipitated with 5 volumes of ice-cold ethanol/ acetone/acetic acid (50/50/0.1, v/v/v) at −20 °C for 2 h. Protein precipitant was centrifuged at 20,000 × g for 1 h. The pellet was washed separately with 1 ml of acetone and 75% ethanol, and redissolved in 600 μL of 6 M guanidine hydrochloride, 100 mM ammonium bicarbonate solution, the protein concentration was determined by Bradford Protein Assay Kit (BOSTER, Wuhan, China) according to the manufacturer’s instructions. The protein concentration of each sample was diluted to 3.00 mg/mL with 6 M guanidine hydrochloride, 100 mM ammonium bicarbonate solution. Cysteine bonds were reduced with 20 mM DTT (dithiothreitol) (Sigma-Aldrich) for 1 h at 56 °C and alkylated with 60 mM IAA (iodoacetamide) (Sigma-Aldrich) for 40 min at room temperature in darkness, and the resulting mixed solution was transferred into ultrafiltration units (10 K, PN: VN01H02, Sartorious). After 30 min of centrifugation at 10,000 × g, the protein adhered to the filter membrane was rinsed 3 times with 0.5 M urea, 100 mM ammonium bicarbonate solution and incubated overnight with trypsin (enzyme-to-protein ratio = 1:50, w/w) in 0.5 M urea, 100 mM ammonium bicarbonate solution at 37 °C. The peptide samples in ultrafiltration units were collected by centrifugation at 10,000 × g for 30 min, and acidified to 1% formic acid. The resulting samples were transferred to 1.5 mL EP tubes for vacuum-dry at 25 °C, the residues were redissolved in loading buffer (65% acetonitrile/2% trifluoroacetic acid/ saturated by glutamic acid) for further TiO2 beads enrichment.
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study
| 100.0 |
The commercial TiO2 beads (GL Sciences, Tokyo, Japan) were used to enrich phosphorylated peptides according to the manufacturer’s instructions. Briefly, 200 μL of loading buffer (65% acetonitrile/ 2% trifluoroacetic acid/ saturated by glutamic acid) was used to equilibrium of TiO2 beads, and then 200 μL of peptide samples in loading buffer were incubated with TiO2 beads (peptides/beads = 1/4, w/w) for 20 min at room temperature. For consecutive incubations, the peptide-beads slurry was incubated and centrifuged, then the supernatant was incubated with another aliquot of freshly prepared TiO2 beads for the next enrichment. The enrichment procedures were repeated two times. The incubated beads were then washed with 800 μL of wash buffer I (65% acetonitrile/0.5% trifluoroacetic acid) and buffer II (65% acetonitrile/ 0.1% trifluoroacetic acid). and the bound peptides were eluted once with the 200 μL elution buffer I (300 mM aqua ammonia/50% acetonitrile) and twice with 200 μL elution buffer II (500 mM aqua ammonia/60% acetonitrile). All the incubation, washing as well as elution procedure, was rotated end-over-end for 20 min at room temperature. The eluates were dried by a vacuum dryer (RE-52AA, Shanghai Zhenjie Instrument Co. Ltd) to obtain the phosphorylated peptides. The enrichment efficiency of commercial TiO2 beads for phosphopeptide was evaluated and the results were shown in Supplementary Figs S3 and S4 and Table S5.
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study
| 100.0 |
The iTRAQ labeling was carried out using an iTRAQ Reagent 4-Plex kit (Applied Biosystems, USA) according to the manufacturer’s protocol. Two sets isotope-labelled samples (one set is Ctr (m/z 114) and Cos (m/z 117); the other is Ctr (m/z 114), Dehy (m/z 115), CD (m/z 116) and VOSL (m/z 117)) were mixed and dried by a vacuum dryer, respectively. The resulting samples were redissolved in 0.1% formic acid (FA), and then were desalted on a C18 ZipTip (P10, Millipore, Billerica) prior to analysis.
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study
| 99.8 |
Dionex ultimate 3000 nano liquid chromatography system (Thermofisher Dionex) was used for online reversed phase chromatographic separation of the phosphopeptide samples prior to nanoelectrospray ion source and mass spectrometric detection. 5 μL of peptide samples (dissolved in 98% H2O/ 2% acetonitrile / 0.1% formic acid) were loaded onto a C18 trap column (0.1 mm × 2 cm, 5 μm, Thermofisher Dionex) using a flow rate of 10 μL/min. The oven temperature was set at 50 °C. Separation of the peptides was performed on a C18 analytical column (75 μm × 150 mm, 3 μm, Thermofisher Dionex) with a flow rate of 400 nL/min. The mobile phase of the binary gradient elution consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid, and separation was performed using the following gradient: 2–8% B over 0–2 min, 8–20% B over 2–75 min, 20–35% B over 75–93 min, 35–80% B over 93–98 min, the composition was held at 80% B for 10 min. All the samples were kept at 4 °C during analysis. The mass spectrometric (MS) data were collected using a maxis impact Q-TOF (Bruker) equipped with an electrospray ionization (ESI) source operating in nanospray positive ion mode. The mass spectrometer was set as follows: Scan mode, full-scan MS and MS/MS scan; MS spectra rate, 3 HZ; MS/MS spectra rate, 5~10 HZ; Acquisition modes, data dependent analysis; Mass and MS/MS range, 50~2500 amu; lockmass, 445 Da; Precursor ion list, 350~1500 m/z; Source Capillary, 1800–1900 v; Dry gas, 2.0 L/min; Dry temperature, 120 °C.
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study
| 99.94 |
The spectral data were imported to “Compass 4.1” software to generate the “.mgf” files, which were subsequently submitted to “Mascot 2.4” software (Matrix Science, London, UK) for protein identification. Database searches of each file were performed using the SwissProt (2013–6) homo sapiens species specific database with the following parameters: peptide tolerance, 10–30 ppm; MS/MS tolerance, 0.05 Da; Quantitation, itraq 4plex; Enzyme, typsin; Max missed cleavages, 2; Fixed modification, carboxymethyl, methylthio of cysteine, iTRAQ 4plex of lysine, and iTRAQ 4plex of the n-terminus; Variable modification, oxidation, phospho of serine, threonine and tyrosine, and iTRAQ 4plex of tyrosine. The identification results of peptides were acceptable when the similarity was >96% and the false discovery rate (FDR) was <1%. The resulting Mascot result files (*.dat) were loaded into the Scaffold Q + S 4.2.0 software (Proteome Software Inc., Portland, OR) for further processing. For relative quantitation, only peptides unique for a given protein were considered, thus excluding those common to other isoforms or proteins of the same family. Proteins were identified on the basis of having at least one peptide with an ion score above 99% confidence. Identified peptides that are unique to a specific protein were used to determine relative quantitation of a protein between the four samples or the two samples, and the intensities of the iTRAQ® reporter ion m/z values (114, 115, 116 and 117) were used to estimate the relative abundances of a particular peptide. For proteins with more than one qualified peptide matches, multiple average peak area ratios were calculated using the peak area ratios of the peptides originating from the same protein58. In the present study, only protein quantification data with relative expression of >1.5 or <0.66 were chosen as differentially expressed proteins, which were analyzed by IPA software version 9.0 (Ingenuity® Systems, California, USA; http://www.ingenuity.com). The functional analysis identified the top pathways and biological functions that were most significant to the data set.
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study
| 100.0 |
Cells were seeded into cell culture dishes (10 cm in diameter), at a density of 1 × 106 cells per dish, and were cultured for 12 h to allow their adhesion to the dish, and then replaced with fresh medium containing two concentration levels, IC30 and IC50, of Cos, Dehy, CD, or VOSL, respectively, whereas the same volume of fresh medium without test compounds were added to the cells as blank control. All culture dishes were incubated for 48 h, and then cells were harvested, resuspended in RIPA lysis buffer, containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail, and centrifuged at 13,000 rpm for 30 min at 4 °C. Protein concentration was determined by bicinchoninic acid (BCA) method, using bovine serum albumin as standard. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in defatted milk (5% in Tris-buffered saline with Tween-20 buffer) at 37 °C for 1 h, and then were incubated with various primary antibodies overnight at 4 °C. Finally, primary antibodies were revealed using horseradish peroxidase-conjugated anti-rabbit antibodies and an ECL chemiluminescence detection system (Pierce). The bands were semi-quantified using Image J software.
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study
| 100.0 |
Cells were seeded into cell culture dishes (10 cm in diameter), at a density of 1 × 106 cells per dish, and cultured for 12 h to allow their adhesion to the dish, and then replaced with fresh medium containing two concentration levels, IC30 and IC50, of Cos, Dehy, CD, or VOSL, respectively, whereas, the same volume of fresh medium without test compounds were added to the cells as a blank control. After incubation for 48 h, cell culture medium was removed and the adherent cells were washed twice with PBS quickly, and then the metabolic activity of adherent cells were quenched with liquid nitrogen, and the intracellular cAMP was extracted and determined as Shao et al.59 with a few modifications. Briefly, 1.0 mL of cold methanol-water (v/v = 4/1) containing 0.1% formic acid was added into the culture dish, and then the adherent cells were scraped, collected and transferred to 2 mL centrifuged tubes, and then ultrasonicated for 3 min (4 s off, 6 s on) in ice bath. After that, the resulting samples were centrifuged at 4 °C for 10 min at 15000 g, and 0.6 mL supernatant was transferred into a 1.5 mL centrifuged tubes, and then dried using nitrogen stream. The residues were resolved with 200 μL acetonitrile-water (v/v = 1/4), and centrifuged at 4 °C for 10 min at 15000 g, 150 μL supernatant was transferred into a glass auto-sampler for LC-MS/MS analysis.
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study
| 100.0 |
Quantification of cAMP was performed on a Waters Acquity UPLC system (Waters® Corporation, MA, USA) using a Waters Acquity BEH C18 column (2.1 × 50 mm2, 1.7 μm) coupled to an AB Sciex Triple QuadTM 6500 mass spectrometer (Applied Biosystems Corporation, MA, USA). The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) and separation on BEH C18 column (2.1 × 50 mm2, 1.7 μm) was achieved under the following gradient: 5–5% B over 0–0.2 min, 5–35% B over 0.2–3.5 min, 35–80% B over 3.5–6 min, 80–100% B over 6–6.5 min, 100–100% B over 6.5–8 min. The flow rate was 0.4 mL/min. The mass spectrometric data were collected using positive ion mode. The source temperature was set at 120 °C with a cone gas flow of 40 L/h, a desolvation gas temperature of 400 °C with a gas flow of 650 L/h, the capillary voltage was set to 5500 V. Collision energy (CE) and declustering potential (DP) were optimized for each standard manually. The external standard method was used for quantification of cAMP, and the contents of cAMP in different groups were adjusted by the total protein concentrations in different groups.
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study
| 100.0 |
Cell cycle was analyzed according to Lohberger et al. report60. Briefly, after incubation with the respective IC10, IC30, IC50 concentrations of Cos, Dehy, CD, or VOSL for 48, cells were harvested by trypsinisation, and then were fixed by ice-cold ethanol (70%) for 10 min at 4 °C. After washing with PBS, the cell pellets were resuspended in propidium iodid (PI) staining buffer (50 μL/mL PI, RNAse A; Beckman Coulter). After 15 min of incubation at 37 °C, cell cycle distribution was analyzed by a FACScalibur System (BD Biosciences) using ModFit software.
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study
| 99.94 |
Annexin V-FITC/PI double staining was employed to quantify the apoptotic rate of MCF-7 cells. Briefly, cells were plated into 6-well plates at 2 × 105 cells per well and cultured for 12 h to allow their adhesion to the dish. After incubation with the respective IC10, IC30, IC50 concentrations of Cos, Dehy, CD, or VOSL for 48 h, cells were harvested by trypsinisation, and then were stained with annexin V-FITC/PI double fluorescence apoptosis detection kit (BD Biosciences) following the manufacturer’s instruction. After incubation for 15 min at room temperature in darkness, the apoptotic cells were detected using a FACScalibur System (BD Biosciences). Compensation was performed by single annexin and PI measurements and data were analyzed by FCS3 express software (De Novo software). Untreated cells were used as a negative control.
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study
| 100.0 |
A total of thirty healthy female BALB/c nude mice (4 weeks old) were provided by Shanghai SLAC Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China), and maintained in specific pathogen-free (SPF) conditions. MDA-MB-231 cells were injected subcutaneously into the right armpit of the nude mice at 4 × 106 cells in 150 μl PBS. After 15 days incubation, three mice with the largest tumors and two mice with the smallest tumors were excluded, and the other twenty-five xenograft mice with tumor size reaching a diameter of about 5 mm were randomly divided into five groups. The Cos, Dehy, CD and VOSL-treated groups were injected intraperitoneally with the corresponding agents at a dose of 20 mg/kg/day, respectively. The negative control (NT) was treated with an equal volume of vehicle. Tumor size was monitored at 0, 3, 10, 17, 24 and 31 days post-treatment, and tumor volume was calculated using the formula: “maximum diameter × minimum diameter2/2. Tumor-bearing mice were sacrificed after 30 times of administrations. Tumors were harvested and weighed. The tissues were cut into consecutive sections for examining the expression of p-AKT, p53, p-14-3-3 and c-Myc by immunohistochemistry. All procedures involving animals were reviewed and approved by the Experimental Animal Ethics Committee of Second Military Medical University, and we confirm that all experiments were performed in accordance with relevant guidelines and regulations.
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study
| 100.0 |
All values are expressed as mean values ± standard error (SE). Student’s unpaired t-test was used to evaluate differences between treated groups and their respective controls. The significance of dose responses was evaluated by repeated measures analysis. IC10, IC30 and IC50 values were calculated using the PASW Statistics 18.0.
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study
| 99.94 |
How to cite this article: Peng, Z. et al. Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway. Sci. Rep. 7, 41254; doi: 10.1038/srep41254 (2017).
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other
| 78.25 |
Symbiotic associations between plants and fungi (mycorrhizae) began about 450 million years ago1. Most mycorrhizal associations are mutualistic, such that the host plant and mycorrhizal fungi exchange nutrients with each other2. However, mycoheterotrophs have evolved a special type of plant–fungi symbiosis in which a plant gets fixed carbon and other nutrients from fungal partners, rather than from photosynthesis3. One of the most interesting characteristics of orchids is the reliance on fungi for seed germination and nutrient absorption, for example, through formation of mycorrhiza with fungi. Over 99% of orchids show partial mycoheterotrophy in which young plants obtain carbon (C) nutrients from fungi prior to the development of green leaves, while adult plants are autotrophic. The extreme type of mycoheterotrophy in orchids is obligate mycoheterotrophy, in which plants are achlorophyllous (lack chlorophyll) throughout their life cycle and therefore fully dependent on fungi for nutrition.
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study
| 65.94 |
Gastrodia elata (Orchidaceae) is an orchid popularly used in traditional Chinese medicine that has a fully mycoheterotrophic lifestyle with highly reduced leaves and bracts in scape, although field guides and systematists often refer to the plants as leafless4,5. During its life cycle, in associates with at least two types of fungi: Mycena for seed germination and Armillaria mellea for plant growth. To obtain nutrition, it forms an association with A. mellea6 and more than 80% of its ~36-month lifespan is spent underground as a tuber (Fig. 1a). These features of the plant are putative adaptions to its obligate mycoheterotrophic lifestyle. G. elata thus offers the possibility of obtaining a valuable insight into the genetic basis of mycoheterotrophy. Here we present a high-quality reference genome assembly of G. elata (Orchidaceae), and use it to investigate the molecular basis of its full mycoheterotrophic life cycle. The observations presented here will be of value for functional ecological studies seeking to understand the mechanisms and evolutionary basis of plant–fungal associations.Fig. 1Gastrodia elata life cycle and gene-family contraction. a The main developmental stages of G. elata. Seeds develop into a protocorm stage without requiring A. mellea (A). The protocorm then differentiates into a corm stage after commencing its association with A. mellea; note that lateral buds develop into juvenile tubers (B). Young and immature tubers (C). Mature tuber with an emergent young scape (D). Scape (stem and inflorescence) of mature plants (E). b Phylogenetic tree of 14 plant species including G. elata. The red dot represents a calibration point determined from the timetree website. c Bar graph of the number of protein-coding genes in each of the species. Single-copy orthologs include common orthologs with one copy in specific species. Multi-copy orthologs include common orthologs with multiple copy numbers in specific species. Other orthologs include genes from families shared in 2–13 species. ‘Eudicot’ clusters with eudicots. ‘Monocots’ clusters with monocotyledonous plants. ‘Orichd’ clusters with G. elata, P. equestris, and D. officinale. ‘Unclustered’ include genes that cannot be clustered into gene families. d Analysis of gene numbers in the genomes of four species for the nucleotide-binding site gene family (NBS), the pathogenesis-related protein (PR) family (Gel G. elata, Peq P. equestris, Dof D. officinale, Aco A. comosus). Numbers in circles represent the number of family members in each genome, and numbers with plus or minus signs indicate, respectively, the number of duplicated or deleted genes. e The plastid genomes of P. equestris (outer circle) and G. elata (inner circle). Red, protein; orange, rRNA; green, tRNA; gray, genes lost from the plastid genome of G. elata
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study
| 99.94 |
Gastrodia elata life cycle and gene-family contraction. a The main developmental stages of G. elata. Seeds develop into a protocorm stage without requiring A. mellea (A). The protocorm then differentiates into a corm stage after commencing its association with A. mellea; note that lateral buds develop into juvenile tubers (B). Young and immature tubers (C). Mature tuber with an emergent young scape (D). Scape (stem and inflorescence) of mature plants (E). b Phylogenetic tree of 14 plant species including G. elata. The red dot represents a calibration point determined from the timetree website. c Bar graph of the number of protein-coding genes in each of the species. Single-copy orthologs include common orthologs with one copy in specific species. Multi-copy orthologs include common orthologs with multiple copy numbers in specific species. Other orthologs include genes from families shared in 2–13 species. ‘Eudicot’ clusters with eudicots. ‘Monocots’ clusters with monocotyledonous plants. ‘Orichd’ clusters with G. elata, P. equestris, and D. officinale. ‘Unclustered’ include genes that cannot be clustered into gene families. d Analysis of gene numbers in the genomes of four species for the nucleotide-binding site gene family (NBS), the pathogenesis-related protein (PR) family (Gel G. elata, Peq P. equestris, Dof D. officinale, Aco A. comosus). Numbers in circles represent the number of family members in each genome, and numbers with plus or minus signs indicate, respectively, the number of duplicated or deleted genes. e The plastid genomes of P. equestris (outer circle) and G. elata (inner circle). Red, protein; orange, rRNA; green, tRNA; gray, genes lost from the plastid genome of G. elata
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study
| 100.0 |
The genome of a G. elata individual was sequenced using a whole-genome shotgun (WGS) approach (Supplementary Table 1). Through K-mer distribution analysis, the genome size was estimated to be 1.18 Gb (Supplementary Fig. 1). The assembly consisted of 3779 scaffolds, with a scaffold N50 of 4.9 Mb (total length = 1061.09 Mb) and contig N50 of 68.9 kb (total length = 1025.5 Mb) (Supplementary Table 2). Overall, 98.51% of the raw sequence reads could be mapped to the assembly, suggesting that our assembly results contained comprehensive genomic information (Supplementary Table 3). Gene region completeness was evaluated by RNA-Seq data (Supplementary Table 4): of the 80,646 transcripts assembled by Trinity, 98.66% could be mapped to our genome assembly, and 94.41% were considered as complete (more than 90% of the transcript could be aligned to one continuous scaffold). The completeness of gene regions was further assessed using CEGMA (conserved core eukaryotic gene mapping approach): 239 of 248 (96.37%) conserved core eukaryotic genes from CEGMA were captured in our assembly, and 217 (87.5%) of these were complete (Supplementary Table 5).
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study
| 100.0 |
Much of the G. elata genome (66.18%) was occupied by transposable elements (TEs). Class I (retrotransposons) and Class II (DNA transposons) TEs accounted for 55.94% and 4.38% of the genome, respectively (Supplementary Table 6). Long terminal repeats (LTRs) formed the most abundant category of TE, with LTR/Gypsy and LTR/Copia occupying 45.04% and 7.10% of the genome, respectively (Supplementary Table 7). Global activity of LTRs was similar between G. elata and Phalaenopsis equestris, while Dendrobium officinale presented a recent burst of LTR activities (Supplementary Fig. 2a). Compared to P. equestris, all LTR families in G. elata had fewer members, except a substantive expansion of del family (Supplementary Table 8 and Supplementary Fig. 2b). Through a combination of ab initio prediction, homology search, and RNA sequence-aided prediction, 18,969 protein-coding genes were predicted in the G. elata genome. Of these genes, 81.6% were functionally annotated (Supplementary Table 9) and 88.69% had detectable transcripts in an RNA-seq analysis of protocorms, tubers (juvenile, immature, and mature tubers), and scapes (Supplementary Table 10). Our transcriptomics analysis revealed that there were 10,548 differentially expressed genes among the five growth stages; these differentially expressed genes clustered into five distinct groups that were representative of the particular stages of growth of G. elata (Supplementary Fig. 3, Supplementary Table 11 and Supplementary Note).
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study
| 100.0 |
Comparison of the sequenced genomes of the orchid species G. elata, P. equestris7, and D. officinale8,9 indicated that they diverged approximately 67 million years ago (Fig. 1b and Supplementary Fig. 4). Two ancient whole-genome duplication (WGD) events are evident in the G. elata genome; these events can also be discerned in the genomes of P. equestris and D. officinale suggesting they occurred prior to the divergence of the three orchid species (Supplementary Fig. 5). The older WGD event might represent the heWGD event10 shared by most monocots, while the younger WGD event were likely shared by all extant orchids and might contribute to the divergence of orchid, as suggested in Apostasia shenzhenica genome11.
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study
| 100.0 |
Compared to P. equestris (29,431 protein-coding genes) and D. officinale (28,910 protein-coding genes), G. elata has a relative small proteome size (18,969 protein-coding genes). The estimated proteome size of G. elata is the smallest theoretical proteome so far identified among angiosperm genomes (Supplementary Table 12). Comparison of G. elata, P. equestris, and D. officinale genes that have functional annotation information revealed global gene set reduction in the G. elata genome. For example, almost all second level gene ontology (GO) categories had fewer genes in G. elata than in the other two species, and 9 of these categories (16.7%) were significantly reduced (Fisher’s Exact test, p < 0.05, Supplementary Fig. 6 and Supplementary Table 13). We also found that several Pfam domain families were significantly reduced in the G. elata genome (Supplementary Table 14). Among the 14 angiosperm used in the phylogenetic analysis, G. elata had the lowest number of gene families; moreover, G. elata had on average the lowest number of genes in each gene family (Fig. 1c, and Supplementary Table 15). This consistently low number of genes and gene families suggests that many gene families have been eliminated from the G. elata genome, and further suggests that many of the remaining gene families have contracted. Gene family expansion and contraction analysis based on maximum likelihood modeling of gene gain and loss confirmed that 3586 gene families had undergone contraction in G. elata, much more compared to the other two orchid genomes (Supplementary Fig. 7 and Supplementary Table 16). A Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis, which assessed 956 orthologous groups with genes present as single-copy in at least 90% of plant genomes12, revealed that 195 (20.4%) highly-conserved genes were missing from the G. elata genome. This rate of absence is much higher than in the genomes of the 13 land species that were included in this analysis (Supplementary Table 17). All of these analyses indicate that G. elata has undergone extensive gene losses, even for genes that were conserved in other plant species that have also undergone extensive lost events.
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The absence of these genes is unlikely to be due to genome assembly problems because 98.66% of the transcripts assembled from transcriptome data could be mapped to the assembly. Another possibility is that several genes were missed due to gene prediction problems. By mapping RNA reads onto the annotated genome, we found that the majority of RNA reads (>86%) from all G. elata tissues could be mapped to annotated exon regions (Supplementary Table 18). This rate of mapping was comparable to that achieved in the well-annotated rice genome and higher than in the P. equestris genome (Supplementary Table 18). Through analysis of gene synteny among G. elata and P. equestris and D. officinale, we detected 2961 gene deletion events in G. elata versus P. equestris, and 3120 gene deletion events in G. elata versus D. officinale (Supplementary Table 19). Further TBLASTN searches of these deleted genes recovered less than 3% of them. Of these genes, fewer than 15% were supported by RNA-seq data (Supplementary Table 19). Both the RNA mapping results and the synteny deletion analysis confirmed that our gene prediction was comprehensive; thus, the possibility of missing gene annotations was low. Finally, PCR amplification of 18 lost genes (atpD, atpG, IhcA, IhcB, psaD, psaF, psaL, psaN, psbO, psbR, psbY, psb27, psb28, petC, petE, ICS, DHAR, and TRX) confirmed that all were absent from the G. elata genome (Supplementary Figs. 8, 9 and Supplementary Table 20). Thus, the global gene losses in G. elata represent evolutionary events, and might be the result of adaption to an obligate mycoheterotrophic lifestyle.
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Both pseudogenizations and genome rearrangements contributed to the gene lost process of G. elata. We found 876 and 1080 pseudogenes in G. elata using P. equestris and D. officinale genes as seeds, respectively (Supplementary Tables 21–24). Through a whole-genome alignment between G. elata and P. equestris, we found 487 genes were lost due to local rearrangements (SV genes, Supplementary Tables 25 and 26). Functional genes in G. elata were located closer to transposable elements than to pseudogenes and SV genes, suggesting that transposable element did not play significant role during the gene lost processes in G. elata (Supplementary Fig. 10). Thus the gene lost processes in G. elata might be dominated by random mutations as we found many pseudogenes in G. elata. Notably, compared with the genomes of P. equestris, D. officinale, A. comosus (pineapple, used in this study as an outgroup) and Arabidopsis thaliana (used in this study as an outgroup), the G. elata genome has a reduced number of genes related to plant resistance to pathogens, such as the NBS (the nucleotide-binding site) gene family, PR (pathogenesis-related) gene family, and genes of antioxidant proteins (Fig. 1d, Supplementary Fig. 11 and Supplementary Table 27). For example, the ICS gene, which is known to function as a primary modulator of salicylic acid-based plant defense responses13, is absent from the G. elata genome, illustrating the loss of a key gene for systemic acquired resistance (Supplementary Figs. 8, 9 and Supplementary Table 27).
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As G. elata does not perform photosynthesis, it was unsurprising that genes enriched for ‘chloroplast’ and ‘plastid’ annotations were strongly represented among the missing genes (Supplementary Table 28). To further investigate the putative functions of missing genes, we examined genes related to the photosynthetic apparatus, namely Photosystem I, Photosystem II, Cytochrome b6f, Cytochrome C6, ATP synthase, and Rubisco14. Of the 35 nuclear genes coding for photosynthetic apparatus proteins (NEP), only 12 were present in the G. elata genome; this is significantly fewer than in A. thaliana, A. comosus, P. equestris, and D. officinale (Supplementary Tables 28, 29). We assume that these genes were non-functional because their full complements of subunits were not present.
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We also sequenced and assembled the plastid genome of G. elata. We found that the plastid genome of G. elata (35,326 bp) was dramatically restructured and reduced in size, in a similar manner to the reduction in gene number observed for the nuclear genome (Fig. 1e), compared to the plastid genomes of P. equestris (148,958 bp)15 and D. officinale (152,221 bp)16, the two other orchid species with sequenced genomes. The plastid genomes of these two species comprise two single-copy regions (a large and a small single-copy region) and the two identical large inverted repeats (IRs) encode 75 and 76 genes, respectively, that are most associated with photosynthesis. The G. elata plastid genome has lost one IR and encodes only 19 protein-coding genes (Fig. 1e), suggesting that G. elata is an ancient mycoheterotroph and that its plastid genome is in the last stage of a ‘degradation ratchet’, i.e., retention and loss of the five core nonbioenergetic genes17,18. Excluding the possibility that these genes were missed by our genome assembly, the transcriptome sequencing analysis indicated that none of the deleted plastid or nuclear encoded genes were expressed in G. elata, while the five core nonbioenergetic genes, trnE, aacD, clpP, ycf1, and ycf2, were moderately to highly expressed in all five stages in G. elata (Supplementary Tables 30, 31). These results clearly show that both the plastid and nuclear genomes of G. elata have lost most of the genes required for photosynthesis, although the highly degraded plastome is still essential for this full mycoheterotroph.
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Although the G. elata genome has clearly undergone extensive gene loss, we found that 430 gene families (19 by a significant margin), containing 1532 genes (184 by a significant margin), showed expansion in G. elata compared to P. equestris, D. officinale, and A. comosus (Supplementary Fig. 7 and Supplementary Tables 32, 33). These genes are enriched for GO terms related to several metabolic processes (Fig. 2a, Supplementary Table 32). We speculate that these expanded genes are related in some way to the functional requirements of the obligate mycoheterotrophic lifestyle of G. elata. We first sequenced and assembled the mitochondrial genome to explore this idea, and the mitochondrial genome G. elata is markedly expanded in size (1339 kb, Fig. 2b) compared to the mitochondrial genomes of most other seed plants19. Thirty-seven protein-coding genes were annotated, and one subunit of mitochondrial ATP synthase, atp4, had two copies in the mitochondrial genome of G. elata and was highly expressed in the cortex layer (Supplementary Table 34). In addition, 36 of the genes had detectable expression in mature tubers (epidermis, cortex, and parenchymal cell) using a tissue-specific qPCR-based analysis (Supplementary Table 34).Fig. 2Gene expansion in G. elata and microbial community analysis. a REViGO semantic similarity scatter plot of Biology Process Gene Ontology terms for expanded genes in G. elata. In semantic spaces, the proximity between circles represents relatedness (similarity) of the GO terms. Similar GO terms are close together in the plot. The axes in the plot have no intrinsic meaning, but were used to measure pairwise similarities between GO terms. Color indicates degree of enrichment for each process presented as the p-value from the hyper-geometric test. b The draft mitochondrial genome of G. elata. Nineteen contigs are manually displayed as a circle, including 12 circular contigs in orange (ornamented with stars) and 7 linear contigs (in blue). The genes are indicated in the middle circle, and are color coded as follows: trn (blue), rrn (light blue), atp (red), and other protein-coding genes (black). The duplicated atp genes and their fragments are detailed in the inner circle. The duplicated genes are suffixed with ‘b’, and the gene fragments are suffixed with ‘fragment’. c Gene expression heat map of the normalized RNA-Seq data for genes encoding the monocot mannose-binding lectin antifungal proteins (GAFP) in G. elata16. The units indicate the expression levels of different gene members of GAFP in the protocorm, juvenile tuber, immature tuber, mature tuber, and scape of G. elata (only shown where the gene expression level RPKM > 1, n = 3). d Venn diagrams showing the number of shared and unique fungal and bacterial operational taxonomic units (OTUs) based on the ITS and 16S sequence analyses in protocorms, juvenile tubers, immature tubers, and mature tubers of G. elata. OTUs showed the composition and abundance of the microbe species, which were defined at 3% dissimilarity
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Gene expansion in G. elata and microbial community analysis. a REViGO semantic similarity scatter plot of Biology Process Gene Ontology terms for expanded genes in G. elata. In semantic spaces, the proximity between circles represents relatedness (similarity) of the GO terms. Similar GO terms are close together in the plot. The axes in the plot have no intrinsic meaning, but were used to measure pairwise similarities between GO terms. Color indicates degree of enrichment for each process presented as the p-value from the hyper-geometric test. b The draft mitochondrial genome of G. elata. Nineteen contigs are manually displayed as a circle, including 12 circular contigs in orange (ornamented with stars) and 7 linear contigs (in blue). The genes are indicated in the middle circle, and are color coded as follows: trn (blue), rrn (light blue), atp (red), and other protein-coding genes (black). The duplicated atp genes and their fragments are detailed in the inner circle. The duplicated genes are suffixed with ‘b’, and the gene fragments are suffixed with ‘fragment’. c Gene expression heat map of the normalized RNA-Seq data for genes encoding the monocot mannose-binding lectin antifungal proteins (GAFP) in G. elata16. The units indicate the expression levels of different gene members of GAFP in the protocorm, juvenile tuber, immature tuber, mature tuber, and scape of G. elata (only shown where the gene expression level RPKM > 1, n = 3). d Venn diagrams showing the number of shared and unique fungal and bacterial operational taxonomic units (OTUs) based on the ITS and 16S sequence analyses in protocorms, juvenile tubers, immature tubers, and mature tubers of G. elata. OTUs showed the composition and abundance of the microbe species, which were defined at 3% dissimilarity
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We next explored how gene expansion in G. elata may have contributed to its association and interactions with fungal microbiota. The monocot mannose-binding lectin antifungal protein family (GAFP) of G. elata contains 20 genes, compared to only 3 in A. comosus and 0 in A. thaliana (Supplementary Table 27). GAFP proteins have been documented to inhibit the growth of both ascomycete and basidiomycete fungal plant pathogens in vitro20. More than 80% of the GAFP genes were highly expressed in protocorms and juvenile tubers, the growth stages that occur before G. elata establishes a stable symbiotic association with A. mellea (Fig. 2c). 4-Hydroxybenzyl alcohol (p-PA), the precursor of the phytoalexin gastrodin is a major phenolic compound of G. elata21. The expression of p-PA biosynthesis genes (e.g., cinnamate 4-hydroxylase, C4H, alcohol dehydrogenase, ADH, hydroxybenzaldehyde synthase, HBS)21 was relatively high in protocorms and juvenile tubers. Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS) metabolite analysis revealed that p-HA is present in G. elata tubers but not in A. mellea hyphae (sampled from tuber, wood, and PDA medium). S-(p-HA)-glutathione was detected in both G. elata tubers and in A. mellea hyphae sampled from tubers (Supplementary Fig. 12, Supplementary Table 35), which putatively suggests that G. elata may transport this phytoalexin to A. mellea and prevent the excessive growth of A. mellea. To investigate the effect of A. mellea on microbial management in G. elata, we performed a 16S ribosomal (rRNA) and rDNA ITS sequencing analysis and found that the diversity of bacterial and microbial species was significantly lower during the protocorm stage than at other growth stages (p < 0.05), which was consistent with the pattern of gene expression of GAFP (Fig. 2c, d, Supplementary Tables 36, 37). This increased diversity of bacteria and fungi during the juvenile tuber to mature tuber periods implies that a compatible mycorrhizal fungus (A. mellea) can affect the structure of the microbial community associated with its host and greatly reduce the antifungal and antibacterial activities as a symbiotic association with A. mellea is established.
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Without the ability to perform photosynthesis, G. elata depends completely on its symbiotic fungus for nutrition. It is thus obvious that the signaling pathways related to the establishment of this symbiotic relationship are crucial for G. elata. Some of the mechanisms underlying the symbiotic interaction between G. elata and A. mellea are similar to those for interactions between other plants and arbuscular mycorrhizal (AM) fungi22. The G. elata genome contains many of the genes known to participate in AM associations (Fig. 3a, Supplementary Table 38). Key genes for biosynthesis and secretion of strigolactone were expanded in G. elata (e.g., carotenoid cleavage dioxygenases, CCDs, for biosynthesis23 and ABC transporters, PDRs, for secretion24) (Supplementary Table 38). It is known that strigolactone can stimulate hyphal branching and development of arbuscular mycorrhizal fungi, which increases the chances of an encounter with a host plant24. We conducted growth assays and confirmed that strigolactone had similar branch-inducing effects in A. mellea (Fig. 3b, Supplemental Fig. 13). The expanded number of genes encoding CCDs and PDRs suggests that G. elata has enhanced its ability to interact with A. mellea to increase the efficiency of the establishment of the symbiotic relationship essential for its nutrition and metabolism. Calmodulin-dependent protein kinase genes of the does-not-make-infections 3 subfamily (DMI3) were also doubled or tripled in G. elata (10 genes) compared to P. equestris (3 genes), D. officinale (5 genes), and A. comosus (4 genes); these genes participate in the Ca2+ spiking process that has been shown to regulate the colonization of plants by fungi25.Fig. 3Strigolactone as a putative signal compound in G. elata in its mycoheterotrophic symbiotic relationship with A. mellea. a Overview of proposed signaling and nutrition transfer in G. elata. The red-labeled genes are expanded in the G. elata genome and P-value of Fisher’s exact test of gene number <0.05 (Supplementary Tables 38, 40, 41). ANT, ANT1-like aromatic and neutral amino acid transporters; ArgJ, glutamate N-acetyltransferase; CCD, carotenoid cleavage dioxygenases; COS, chitooligosaccharides; DMI3, does-not-make-infections 3 subfamily; LOS, lipochitooligosaccharides; LysM, LysM-receptor-like kinases; PDR, ABC transporter. P1, p-value of Fisher’s exact test of gene number in G. elata genome compared to P. equestris, D. officinale, A. comosus, and A. thaliana; P2, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. thaliana; P3, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. comosus; P5, p-value of Fisher’s exact test of gene number in G. elata, P. equestris, and D. officinale genome compared to A. comosus and A. thaliana. b Branching of A. mellea hyphae was significantly promoted after strigolactone treatment (Supplementary Fig. 13). Scale bar was 5 mm. c Cross sections and micrographs of immature G. elata tubers in association with A. mellea (A, epidermis; B, cortex; C, inner parenchyma cells, Supplementary Fig. 14). The black scale bar on the left was 100 μm and the white one on the right was 20 μm
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Strigolactone as a putative signal compound in G. elata in its mycoheterotrophic symbiotic relationship with A. mellea. a Overview of proposed signaling and nutrition transfer in G. elata. The red-labeled genes are expanded in the G. elata genome and P-value of Fisher’s exact test of gene number <0.05 (Supplementary Tables 38, 40, 41). ANT, ANT1-like aromatic and neutral amino acid transporters; ArgJ, glutamate N-acetyltransferase; CCD, carotenoid cleavage dioxygenases; COS, chitooligosaccharides; DMI3, does-not-make-infections 3 subfamily; LOS, lipochitooligosaccharides; LysM, LysM-receptor-like kinases; PDR, ABC transporter. P1, p-value of Fisher’s exact test of gene number in G. elata genome compared to P. equestris, D. officinale, A. comosus, and A. thaliana; P2, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. thaliana; P3, p-value of Fisher’s exact test of gene number in G. elata genome compared to A. comosus; P5, p-value of Fisher’s exact test of gene number in G. elata, P. equestris, and D. officinale genome compared to A. comosus and A. thaliana. b Branching of A. mellea hyphae was significantly promoted after strigolactone treatment (Supplementary Fig. 13). Scale bar was 5 mm. c Cross sections and micrographs of immature G. elata tubers in association with A. mellea (A, epidermis; B, cortex; C, inner parenchyma cells, Supplementary Fig. 14). The black scale bar on the left was 100 μm and the white one on the right was 20 μm
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After A. mellea colonizes G. elata, fungal growth is restricted to its cortex layer (Fig. 3c, Supplemental Fig. 14). We performed a tissue-specific qPCR-based analysis of 10 genes in G. elata tubers and found that PDR transcripts, which mediate secretion of strigolactone to the extracellular space, were highly abundant in the cortex layer (Supplementary Fig. 15). This finding suggests that G. elata may preferentially guide A. mellea to colonize its cortex layer. Similarly to ATP synthases, we found that some glycoside hydrolases from gene families that have expanded in the G. elata genome were also highly expressed in the cortex layer, supporting the idea that A. mellea hyphal walls are digested in the cortex layer of G. elata tubers (Supplementary Table 39). The expanded endo-β-1,4-d-xylanase and β-glucosidase may have become neofunctionalized to cleave fungal glycan substrates during the digestion of hyphal walls of A. mellea (Fig. 3a, Supplementary Table 40)26,27.
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Given that the ANT1-like aromatic and neutral amino acid transporters (ANT)28 are known to translocate arginine (Arg), which is a key component in nitrogen translocation in arbuscular mycorrhizal fungi29, it seems likely that Arg in G. elata is related to mycoheterotrophic symbiosis (Fig. 3a, Supplementary Table 41). It is known that arginases can hydrolyze Arg into urea in mycelia, which is further hydrolyzed to ammonium and carbonic acid by ureases30. Although P. equestris, D. officinale, A. comosus and A. thaliana have only one copy of glutamate N-acetyltransferase (ArgJ), an enzyme of the arginine biosynthesis pathway31, G. elata has three copies (Supplementary Fig. 11 and Supplementary Table 41). The number of genes encoding ureases is drastically expanded in G. elata (9 genes) compared with P. equestris (2 genes), D. officinale (2 genes), A. comosus (1 gene), and A. thaliana (1 gene) (Supplementary Table 41). This suggests that urea metabolism might be an important source of nitrogen for G. elata (Fig. 3a).
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The extensive deletion and expansion of genes, especially the global reduction of gene complements in almost all functional categories in the G. elata genome, provides a powerful example of how a plant with a fully heterotrophic life cycle has made use of genome plasticity to achieve extensive neo-functionalization and gene loss. Our results establish a unique opportunity for researchers to understand how plants that have abandoned photosynthesis continue to persist and thrive.
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The experimental materials of Gastrodia elata were harvested from Xiaocaoba in Yunnan Province (latitude 27.79°N longitude 104.24°E) located in the southwestern China. Genome sequencing and assembly was done on the scape of beige-scape G. elata. Five transcriptomes were sequenced from five different G. elata tissues (protocorm, juvenile tuber, immature tuber, mature tuber, scape). Four different G. elata tissues (protocorm, juvenile tuber, immature tuber, mature tuber) were collected to investigate the diversity of microbial communities. High-quality genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit.
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Multiple paired-end and mate-pair libraries were constructed with a spanning size that ranged from 180 bp to 20 kb. Sequencing was conducted on an Illumina HiSeq 2500 platform. In total, 179.1 Gb raw sequencing reads were produced (Supplementary Table 1). Raw sequencing reads were subjected to filtering to remove (1) low quality reads with low quality bases (>50% bases with Q-value ≤8); (2) reads with Ns >10% of the read length; (3) reads with adapter contamination; and (4) duplicated reads caused by PCR during library construction. Filtered data were assembled using ALLpaths-LG (version 44080)32, where overlapping paired-end reads with an insert size of 230 nucleotides were used as fragment libraries, and all other libraries (>230 nucleotide insert size) were used as jumping libraries. The Allpaths-LG assembly was run with default settings, then a gap filling step was carried out using GapCloser based on the paired-end information of the paired-end reads that had one end mapped to the unique contig and the others located in the gap region (http://sourceforge.net/projects/soapdenovo2/files/GapCloser).
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To evaluate the completeness of the assembly and the uniformity of the sequencing, all the paired-end reads were mapped to the assembly using BWA33. The mapping rate was 98.51% and the genome coverage was 99.84%. This result suggested that our assembly results contained almost all the information in the reads (Supplementary Table 3). Gene region completeness was evaluated from the scape tissue, of 80,646 transcripts assembled by Trinity34, 98.66% could be mapped to our genome assembly, and 94.41% were considered as complete (more than 90% of the transcript could be aligned to one continuous scaffold). CEGMA35 (Core Eukaryotic Genes Mapping Approach) defined a set of conserved protein families that occur in a wide range of eukaryotes, and identified their exon–intron structures in a novel genomic sequence. Through mapping to the 248 core eukaryotic genes, a total of 239 genes with a ratio of 96.37% were found in G. elata (Supplementary Tables 5). Genome completeness was also assessed using BUSCO gene set analysis version 2.013 which includes a set of 956 single-copy orthologous genes specific to Plantae.
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A combined strategy based on homology alignment and de novo search was used to identify repeat elements in the G. elata genome. For de novo prediction of transposable elements (TEs), we used RepeatModeler (http://www.repeatmasker.org/RepeatModeler.html), RepeatScout36, and LTR-Finder37 with default parameters. For alignment of homologous sequences to identify repeats in the assembled genome, we used RepeatProteinMask and RepeatMasker (http://www.repeatmasker.org) with the rebase library38. Transposable elements overlapping with the same type of repeats were integrated, while those with low scores were removed if they overlapped more than 80 percent of their lengths and belonged to different types (Supplementary Table 7).
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Intact Long terminal-repeat retrotransposons (LTR) were identified by searching the genomes of G. elata, D. officinale and P. equestris with LTRharvest39 from Genome Tools v1.5.1. The candidate sequences were filtered by two-step procedure to reduce false positives. First, LTRdigest40 was used to identify the primer binding site (PBS) motif based on the predicted tRNA sequences from tRNAscan-SE41, and only elements contained PBS were retained; then protein domains (pol, gag and env) in candidate LTR retrotransposons were identified by searching against HMM profiles collected by Gypsy Databas (GyDB)42. Elements contained gag domain, protease domain, reverse transcriptase (RT) domain and integrase domain, which were considered as intact. Second, families of these intact LTR retrotransposons were clustered using the previously described method43. Finally, LTRs that did not contain protein domains or that belonged to families with less than 5 members were discarded. The EMBOSS program distmat44 was used to estimate LTR divergence rates between the 5′- and 3′- LTR sequences of the intact LTRs (Supplementary Fig. 2).
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Gene prediction was conducted through a combination of homology-based prediction, ab initio prediction and transcriptome-based prediction methods. Protein repertoires of plants including A. comosus10, Amborella trichopoda45, Arabidopsis thaliana (phytozomev10), Brachypodium distachyon (phytozomev10), D. officinale9, O. sativa (phytozomev10), P. equestris7, Vitus vinifera (phytozomev10), Sorghum bicolor (phytozomev10) and Zea mays (phytozomev10) were downloaded and mapped to the G. elata genome using TBLASTN (E-value ≤ 1e−5). The BLAST hits were conjoined by Solar software46. GeneWise (version 2.4.1)47 was used to predict the exact gene structure of the corresponding genomic region on each BLAST hit. Homology predictions were denoted as “Homology-set”. RNA-seq data derived from protocorm, juvenile tuber, immature tuber, mature tuber, and scape (Fig. 1a) were assembled by Trinity (version 2.0) 41. The Trinity assembly included 183,515 contigs with an average length of 592 bp. These assembled sequences were aligned against the G. elata genome by PASA (Program to Assemble Spliced Alignment)48. Valid transcript alignments were clustered based on genome mapping location and assembled into gene structures. Gene models created by PASA were denoted as PASA-T-set (PASA Trinity set). Besides, RNA-seq reads were directly mapped to the genome using Tophat (version 2.0.8)49 to identify putative exon regions and splice junctions; Cufflinks (version 2.1.1) was then used to assemble the mapped reads into gene models (Cufflinks-set). Augustus (version 2.5.5)50, GeneID (version)51, GeneScan (version 1.0)52, GlimmerHMM (version 3.0.1)53, and SNAP (version)54 were also used to predict coding regions in the repeat-masked genome. Of these, Augustus, SNAP and GlimmerHMM were trained by PASA-H-set gene models. Gene models generated from all the methods were integrated by EvidenceModeler (EVM)48. Weights for each type of evidence were set as follows: PASA-T-set > Homology-set > Cufflinks-set > Augustus > GeneID = SNAP = GlimmerHMM = GeneScan. The gene models were further updated by PASA2 to generate UTRs, alternative splicing variation information, which generated 26,872 gene models. Gene models only supported by ab initio evidence were filtered out. To reduce the possibility of missing and poorly annotated genes, we invested additional effort in annotating some gene families that could be missed by automated genome annotation, such as NBS-encoding genes. In total, 1943 protein sequences containing an NB-ARC domain were searched against the G. elata genome using TBLASN with a threshold of 1e−5. All BLAST hits in the genome, together with 5000 bp flanking regions on both sides, were annotated by the GeneWise program. The resulting predictions were surveyed to verify whether they encoded NBS or LRR motifs using Pfam. We also focused on other genes, such as those related to photosynthesis, and transporter, and these were manually annotated through a combination of BLAST search and motif verification. Ultimately, a comprehensive non-redundant reference gene set was produced that contained 18,969 protein-coding gene models. Functional annotation of the protein-coding genes was carried out using BLASTP (E-value cut-off 1e−05) against two integrated protein sequencing databases, SwissProt and TrEMBL55. Protein domains were annotated by searching against InterPro (Version 5.16)56 and Pfam (Version 3.0) database57, using InterProScan (version 4.8) and HMMER (version 3.1b1) (http://hmmer.janelia.org), respectively. The GO terms for genes were obtained from the corresponding InterPro or Pfam entry. The pathways in which the genes might be involved were assigned by BLAST against the KEGG databases (release 20150831)58 with the E-value cut-off of 1e−05.
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Pseudogenes in the G. elata genome were identified by searching against G. elata intergenic regions using D. officinale or P. equestris protein sequences as the seed sequences (TBLASTN, E-value cut-off 1e−5). Before the BLAST search, regions of the 18,969 true genes were masked. The BLAST hits were conjoined by Solar software. GeneWise was used to predict the pseudogene structures with the ‘-pseudo’ parameter. Pseudogenes were then classified by PseudoPipe59. The PseudoPipe program applies a set of sequence identity and completeness cut-off to report a final set of good-quality pseudogene sequences. We used the following cutoffs: amino acid (AA) sequence identity >30% and match length >50 AA to filter out false positives. GeneWise results that fulfilled the cut-off criteria were denoted as high-confidence pseudogenes. High-confidence pseudogenes were then assigned to three categories. (1) Processed/retrotransposed pseudogenes (PSSDs), which formed through retrotransposition. Retrotransposition occurred by reintegration of a cDNA, a reverse transcribed mRNA transcript, into the genome at a new location. (2) Duplicated pseudogenes (DUPs), which formed through gene duplication, following by decay of genes, include frameshifts or premature stop codons. (3) Pseudogenic fragments (FRAGs), which were fragments that have high-sequence similarity to known proteins, but were too decayed to be reliably assessed as processed or duplicated. We used the following criteria to classify PSSDs, DUPs, and FRAGs: (i) PSSDs, exon number = 1, 0.7 < align ratio ≤ 0.95, 0.3 ≤ identity ≤ 0.95; (ii) DUPs, exon number > 1, 0.3 ≤ identity ≤ 0.95, and existing insertion, deletion, termination, or frameshift; (iii) FRAGs, exon number = 1, align ratio < 0.7, 0.3 ≤ identity ≤ 0.95.
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Whole protein-coding gene repertoires from 14 plant genomes including G. elata, A. comosus 10, A. trichopoda 45, O. sativa (phytozome v10), Z. mays (phytozomev10), D. officinale9, P. equestris7, Elaeis oleifera60, A. thaliana (phytozomev10), V. vinifera (phytozomev10), Populus trichocarpa (JGI), Glycine max (phytozomev10), Picea abies61, Physcomitrella patens (ASM242v1) were used to construct a global gene family classification. To remove redundancy caused by alternative splicing variations, we retained only gene models at each gene locus that encoded the longest protein sequence. To exclude putative fragmented genes, genes encoding protein sequences shorter than 50 amino acids were filtered out. All-against-all BLASTp was employed to identity the similarities between filtered protein sequences in these species with an E-value cut-off of 1e−7. The OrthoMCL62 method was used to cluster genes from these different species into gene families with the parameter of “-inflation 1.5”.
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Protein sequences from 74 single-copy gene families were used for phylogenetic tree reconstruction. MUSCLE63 was used to generate multiple sequence alignment for protein sequences in each single-copy family with default parameters. Then, the alignments of each family were concatenated to a super alignment matrix. The super alignment matrix was used for phylogenetic tree reconstruction through maximum likelihood (ML) methods. Before ML reconstruction, we used ProtTest64 to select the best substitution models. The JTT + I + G + F model was selected as the best-fit model, and RAxMLwas used to reconstruct the phylogenetic tree65.
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Divergence time between 14 species was estimated using McMctree in PAML66 with the options ‘correlated molecular clock’ and ‘JC69’ model. A Markov Chain Monte Carlo analysis was run for 20,000 generations, using a burn-in of 1000 iterations. Five calibration points were applied in the present study (Fig. 1): P. equestris and D. officinale divergence time (47~52.9 million years ago) 67, O. sativa and Z. mays divergence time (24–84 million years ago)68,69, A. thaliana and P. trichocarpa divergence time (65–89 million years ago) 70,71, P. trichocarpa and G. max divergence time (56–89 million years ago)56,57, and, root of land plants (407–557 million years ago) 57.
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Expansion and contractions of orthologous gene families were determined using CAFÉ 2.2 (Computational Analysis of gene Family Evolution). The program uses a birth and death process to model gene gain and loss over a phylogeny. Large changes in gene family size in a phylogeny were tested by calculating p-values on each branch using the Viterbi method with a randomly generated likelihood distribution. This method calculates exact p-values for transitions between the parent and child family sizes for all branches of the phylogenetic tree. Enrichment of Gene Ontology terms for G. elata expanded gene families were summarized and visualized using REVIGO (small list, similarity (0.5), SimRel similarity measure).
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The expanded and contracted families focused on in this study were confirmed using Fisher’s exact test. For each gene family, we compared the gene count of the tested family in G. elata (copy number of the tested family as numerator, total number of genes of the whole genome as denominator) versus the frequency in D. officinale8,9, P. equestris7, A. comosus10, and Arabidopsis thaliana (phytozomev10). In addition, phylogenetic trees were constructed for each family to confirm gene gain or loss events. The extreme case of gene lost was that one gene was absent in the G. elata genome. To avoid false positive gene absence events caused by missing gene annotations, a TBLASTN search against the G. elata genome was carried out using protein sequences derived from other plant genomes.
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study
| 100.0 |
Total genomic DNAs were extracted by hexadecyl trimethyl ammonium bromide (CTAB) method from G. elata, A. thaliana, A. comosus, and D. officinale. PCR was carried out using SpeedSTARTM HS DNA Polymerase (TaKaRa, Japan) and specific gene primers (Supplementary Table 20). PCR products were verified by agarose gel electrophoresis (Supplementary Figs. 8 and 9).
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study
| 99.94 |
A homolog search within the G. elata genome was performed using BLASTP (E-value < 1e−7), and MCscanX was used to identify syntenic blocks within the genome. For each gene pair in a syntenic block, the 4DTv (transversion substitutions at fourfold degenerate sites) distance was calculated, and values of all gene pairs were plotted to identify putative whole-genome duplication events in G. elata.
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study
| 100.0 |
Proteins of G. elata, P. equestris and D. officinale were aligned using the BLASTp algorithm (E-value < 1e−7). Alignments with matches of at least 30% identity and coverage higher than 30% were retained for comparison. The best reciprocal BLAST pairs between different genomes were extracted as putative orthologous gene pairs. Then, using gene location information in each species, we identified micro-synteny gene blocks between G. elata and the other two orchids. Putative gene loss events were traced from the synteny table using the flanking gene method. Given three genes A, B, and C in order, if gene A and C were presented as collinear orthologs in two genomes, but B was missed in one of the genome (for example, G. elata), then gene B was denoted as a possible lost gene in G. elata. To avoid false positives due to the failure of gene annotation, the G. elata intergenic genomic sequence between A and C was extracted, and a GeneWise prediction in this intergenic region was carried out using the B protein sequence from P. equestris as seed. If the predicted protein could be aligned to the seed protein with coverage >70%, and did not contain frameshift or premature stop codon mutations, this gene loss event was defined as a false positive and filtered out (Supplementary Table 19).
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study
| 100.0 |
Total genomic DNA was extracted using a modified CTAB from silica-dried tissues of G. elata (Supplementary Table 42). The DNA was sheared to 500 bp, and sequencing libraries were generated using the NEBNext Ultra DNA Library Prep Kit (according to the manufacturer’s protocol) for sequencing on an Illumina Hiseq 2500 at the State Key Laboratory of Systematics and Evolutionary Botany, Chinese Academy of Sciences. The raw reads were filtered using NGSQCTOOLKIT v 2.3.3. The cleaned reads were mapped to Calanthe triplicata in GENEIOUS 9.0 (Biomatters, Inc., Auckland, New Zealand; http://www.geneious.com). Used reads were exported and assembled using SOAPDENOVO2. The plastid sequences were extracted from the total contigs using BLASTN 2.2.29+ and the C. triplicata plastome (GenBank ID: NC_024544) as subject sequence. The finished plastome scaffolds were reoriented according to the C. triplicata reference plastome. The boundaries of IRs were determined by BLAST, and finished manually. The plastomes of G. elata were determined using DOGMA with an e-value of 5, a 60% cut-off for protein-coding genes and 80% cut-off for tRNAs; the GENEIOUS annotation tool was used to determine the plastomes of C. triplicata and Oncidium Gower Ramsey (GenBank ID: NC_014056.1) as references. Linear plastome maps were drawn using OGDRAW.
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study
| 100.0 |
Mitochondria were isolated from all G. elata tissues except the rhizome using previously described centrifugation methods72. A modified CTAB method was used to extract mt-DNAs19. The purified mt-DNAs were sequenced on an Illumina Hiseq 2500 to generate 100 bp paired-end reads at the State Key Lab of Systematic and Evolutionary Botany, Chinese Academy of Sciences (Beijing).
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study
| 99.94 |
Eleven million raw reads were generated from sequencing and trimmed using Trimmomatic v0.35 to produce low quality reads. All sequenced plant mitochondrial genomes were downloaded from NCBI and used as a local blast database. To minimize the possibility of contaminated reads from plastid or nuclear genomes, filtered reads were first mapped to the local database and mapped reads were subsequently imported into Geneious v10.1.3 (Biomatters, Inc., Auckland, New Zealand; http://www.geneious.com) for initial assembly. The contigs generated by the initial assembly were used as seeds for further iterative mapping and extension processes. Velvet and Geneious were alternatively used during assembly with multiple combinations of k-mer lengths.
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study
| 99.94 |
In most cases, the extension process of the assembly worked well. In particular, when the head and tail of a contig had an overlapping region and could not be further extended, this contig could be reasonably connected into a circle. Although several circles were produced during assembly, some problems did arise in the extension process. For example, some contigs were displayed as single lines because their boundaries were too difficult to determine due to poly structures or repeats in the mitogenome. The final assembled results were verified by remapping and some ambiguous regions with low coverage were further checked by PCR. Overall, 19 contigs with a total length of 1,340,105 bp were assembled including 12 circles (ranging from 13.5 to 120.6 kb) and 7 single lines (837,015 bp).
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study
| 100.0 |
The assembled contigs were firstly annotated by NCBI-BlastN based on the local database with an e-value < 1e−6. Then, the boundaries of each gene were confirmed by Mitofy and exported as Sequin formatted files. tRNA genes were further predicted by tRNAscan (http://lowelab.ucsc.edu/tRNAscan-SE/)(Supplementary Table 43).
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study
| 99.94 |
The paired-end reads for protocorm, juvenile tuber, immature tuber, mature tuber, scape samples were mapped to the G. elata genome using TopHat. The total numbers of aligned reads were normalized by gene length and sequencing depth for an accurate estimation of expression level. We used these normalized read counts (RPKM) as the expression level for each gene. Then, DESeq was used to identify differentially expressed genes (Supplementary Tables 44-58). A repeated bisection method and a top–down hierarchical clustering algorithm in gCLUTO (http://glaros.dtc.umn.edu/gkhome/cluto/gcluto/overview) were used to generate the expression profiles of all differently expressed genes.
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study
| 100.0 |
Four different G. elata tissues (epidermis, cortex, parenchymal cell A and B) were collected from three mature tuber samples. Total RNAs were extracted using TRIzol® Reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The concentrations and purities of the total RNAs were assessed by a spectrophotometric analysis at 260 and 280 nm. One μg of total RNA was reverse transcribed at 42 °C using TransScript® Reverse Transcriptase (TransBionovo Co, China) and Oligo(dT)18 according to the manufacturer’s recommendations. Prior to use in qPCR, cDNA was diluted 1:5 with H2O.
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study
| 99.94 |
The qPCR reactions were performed in duplicate for each condition using the KAPA SYBR® FAST qPCR Master Mix (KapaBiosystems, USA) and LightCycler® 480 Real-Time PCR System (Roche, Switzerland). Each reaction consisted of 20 μL containing 1 μL of cDNA and 200 nM of each primer (Supplementary Tables 59 and 60). The cycling conditions were: denaturation at 95 °C for 3 min; followed by 45 two-segment cycles of amplification at 95 °C for 10 s, and 60 °C for 30 s in which fluorescence was automatically measured, and one three-segment cycle of 95 °C for 5 s, 65 °C for 1 min, and 95 °C for 30 s. The baseline adjustment method of the LightCycler® 480 software was used to determine the Ct in each reaction. β-actin was selected as the internal control and the expression levels of tested genes were determined using the comparative Ct (2−ΔΔCt) method.
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study
| 99.94 |
Four different G. elata tissues (protocorm, juvenile tuber, immature tuber, mature tuber) were collected and total genomic DNAs were extracted using hexadecyl trimethyl ammonium bromide (CTAB). The 16S V4 and ITS1 genes in all sample were amplified using the universal primers 515F-806R and ITS5-1737F with a barcode as a marker for distinguishing samples. The PCR was performed with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, UK). PCR products were mixed in equidensity ratios. Then, the mixed PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s recommendations and index codes were added. The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina HiSeq 2500 platform and 250 bp paired-end reads were generated.
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study
| 100.0 |
High-quality sequences were clustered into OTUs defined at 97% similarity. These OTUs were applied for diversity, richness and rarefaction curve analyses using MOTHUR. Taxonomic assignments of OTUs that reached the 97% similarity level were made using the QIIME (quantitative insights into microbial ecology) software package through comparison with the SILVA, Greengene, and RDP databases. Venn diagrams were generated to identify the mutual and specific taxons between groups using R software (http://www.r-project.org/).
|
study
| 99.94 |
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