To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers. Approximately 90% of these 385 genes are conserved in humans, and one-third of the conserved genes have been implicated in human disease. Furthermore, many have roles in developmental processes, which suggests that mitochondrial function and their response to stress are defined during development and maintained throughout life. Our dataset primarily contains mitochondrial enhancers and non-mitochondrial suppressors of UPRmt, indicating that the maintenance of mitochondrial homeostasis has evolved as a critical cellular function, which, when disrupted, can be compensated for by many different cellular processes. Analysis of the subsets “non-mitochondrial enhancers” and “mitochondrial suppressors” suggests that organellar contact sites, especially between the ER and mitochondria, are of importance for mitochondrial homeostasis. In addition, we identified several genes involved in IP3 signaling that modulate UPRmt in fzo-1 mutants and found a potential link between pre-mRNA splicing and UPRmt activation.Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPR Caenorhabditis elegans, fusion of the outer and inner mitochondrial membrane (OMM and IMM) is facilitated by FZO-1MFN1,2 (OPA1 (DRP1 (Saccharomyces cerevisiae (Dnm1p) has been shown to form constricting spirals around mitochondria , instead of being imported into mitochondria, translocates from the cytosol to the nucleus, where it activates a broad transcriptional program . Moreover, UPRmt has been shown to act in a cell non-autonomous way, and once activated in a certain tissue can result in a systemic response (Mitochondria are important for cellular adenosine triphosphate (ATP) production, iron–sulfur-cluster biogenesis, lipid metabolism and apoptosis, and therefore, mitochondrial homeostasis is tightly regulated by several quality control mechanisms . Moreoveitophagy . How theresponse .mt in fzo-1(tm1133) mutants and identified 299 suppressors and 86 enhancers. We analyzed this dataset using bioinformatic tools, such as GO enrichment analysis, gene network analysis and analysis of transcription factor (TF) binding sites in promotors of candidate genes. Furthermore, we determined the specificities of the candidates identified with respect to their ability to modulate UPRmt using secondary screens. Finally, we identified the C. elegans ortholog of the mammalian genes Miga1 and Miga2, which have been implicated in mitochondrial fusion, and demonstrate that the loss of the C. elegans ortholog leads to mitochondrial fragmentation and the induction of UPRmt.In this study, we performed a genome-wide RNAi screen to identify regulators of UPRC. elegans strains were cultured as previously described (spg-7(ad2249) (fzo-1(tm1133) ; eat-3(ad426) (drp-1(tm1108) ; bcSi9 (Phsp-6::gfp::unc-54 3’UTR) (miga-1(tm3621) . In addition, the following multi-copy integrated transgenes were used: zcIs9 (Phsp-60::gfp::unc-54 3’UTR), zcIs13 (Phsp-6::gfp::unc-54 3’UTR) (bcIs78 (Pmyo-3::gfpmt) (escribed . BristolC. elegans protein-coding genes. For the primary and secondary screens with the multi-copy zcIs13 transgene in the fzo-1(tm1133), drp-1(tm1108), eat-3(ad426), or spg-7(ad2249) background, RNAi clones were cultured overnight in 100 µl of LB containing carbenicillin (100 μg/mL) in a 96 well plate format at 37°C and 200 rpm. 10 µl of each RNAi culture was used to seed one well of a 24 well RNAi plate containing 0.25% Lactose (w/v) as described previously (fzo-1(tm1133) and spg-7(ad2249) allele, and 2 L4 larvae of drp-1(tm1108) were transferred to each well of the RNAi plates. The F1 generation was scored by eye for fluorescence intensity of the Phsp-6mtHSP70gfp reporter after 4–12 days and compared to worms of the respective genotype on the negative control sorb-1(RNAi).RNAi by feeding was performed using the updated “Ahringer” RNAi library , which crps-1(RNAi) was diluted 1:1 with either empty vector RNAi (L4440) or kgb-1(RNAi).For double-RNAi experiments , RNAi clones were cultured as described above but experiments were conducted in three independent experiments using RNAi plates containing 6 mM IPTG. hsp-6mtHSP70gfp reporter were picked from the wells and inoculated in 100 µl of LB containing carbenicillin (100 μg/mL) in a 96 well plate format and cultured overnight at 37°C and 200 rpm. Glycerol stocks of these overnight cultures were prepared the following day by adding 100 µl of LB containing 30% glycerol and frozen at −80°C. After all RNAi clones of the library were tested, the 657 identified candidates were retested at least three times in duplicates for verification of the observed phenotype. The RNAi clones that reproduced the suppression or enhancement phenotype at least three out of six times were considered as verified candidates.For the primary screen, all RNAi clones of the library were tested once. Bacterial RNAi clones that enhanced or suppressed the PL4440F and L4440R. To remove excessive primers and nucleotides, PCR products were treated with ExoSAP-IT™  according to manufacturer’s protocol. After PCR clean-up, samples were sent for sequencing using L4440F primer.The 385 verified RNAi clones were sequenced. For this, colony PCRs were performed directly from the glycerol stocks using the primers L4440 F 5′-TGGATAACCGTATTACCGCC-3′L4440 R 5′-GTTTTCCCAGTCACGACGTT-3′According to our sequencing results, seven of the RNAi clones covered two genes. These are indicated in column B (“Sequence”) in Supplementary Table S1. These RNAi clones were assigned to the GO group of the gene, which was predominantly covered by our sequencing result and all subsequent analysis were carried out using this gene.drp-1(tm1108), eat-3(ad426), and spg-7(ad2249) mutant backgrounds.Subsequently, the verified and sequenced clones were rescreened in technical duplicates in three independent experiments in the secondary screens in https://intermine.wormbase.org    for compC. elegans genome as a background list. Using an EASE score from the modified fisher-exact test, the clustering algorithm groups genes based on their association in GO categories and assigns a significance value to the group (In search of enriched gene ontology (GO) terms, we used the DAVID tool -induced UPRmt) with WBGene-IDs of the identified candidate genes were used to search in the Transfac database  in specific tissues and at specific times during development can lead to both systemic activation of UPRmt and longevity .In the clustered gene enrichment analysis, we found that the majority of both suppressors and enhancers are associated with at least one of the following GO-terms: “nematode larval development,” “embryo development ending in birth or egg hatching” or “reproduction” (Supplementary Table S2). It has been shown that reducing the functions of some genes encoding components of the ETC [fzo-1(tm1133)-induced UPRmt most probably by inducing autophagy thereby causing changes in lipid metabolism  of the suppressors  and inclmt or simply reduces the expression of the Phsp-6mtHSP70gfp reporter. A previous study also identified many genes related to ribosome biogenesis and cytosolic translation in a screen for suppressors of paraquat-induced UPRmt  upon attenuation of cytosolic translation by rpl-36(RNAi). In contrast, they showed that Pgst-4 HPGDSgfp was slightly hyperactivated  and which presumably also compromises cytosolic translation, results in reduced expression of a control reporter that is unrelated to other stress responses, Pges-1GES2gfp  in C. elegans . Thus, attenuation of cytosolic translation may activate cSADDs through KGB-1MAPK10, thereby preventing UPRmt induction in fzo-1(tm1133) mutants.Moreover, we assigned a substantial part of the suppressors to the groups “RNA processing” (38), “transcription” (35), and “translation” (27) . Hence, ctivated . We rece elegans , derepremt in fzo-1(tm1133). Disruption of these processes has also previously been shown to induce UPRmt in wild type .In summary, the GO enrichment analysis revealed that depletion of the majority of candidates in our dataset may modulate UPRhttps://intermine.wormbase.org) (https://ihg.gsf.de/ihg/mitoprot.html)   for compmt are three genes  with yet unknown functions, which lack orthologs in other systems. ORC-1ORC1 is a component of the origin recognition complex and plays a role in DNA replication   and therefore enhance UPRmt. NHR-209HNF4A,G is orthologous to Hepatocyte Nuclear Factor 4α (HNF4A) and belongs to the family of nuclear hormone receptors, a class of cofactor and ligand-inducible TFs that regulate various cellular processes, including metabolism, development, and homeostasis . Moreover, we identified cpna-3CPNE5,8,9, an ortholog of mammalian copine family members, a class of calcium dependent phospholipid binding proteins with roles in intracellular signaling and membrane trafficking , which is involved in the formation of coat protein complex I (COPI) vesicles. COPI vesicles play a central role in the secretory pathway and are required for the retrieval of lipids and proteins from the Golgi apparatus and the subsequent retrograde transport of these lipids and proteins to the ER  and, consequently, to the activation of UPR in the ER (UPRER) (mt induction in fzo-1(tm1133) animals upon copd-1(RNAi) may be due to alterations in one of the above-mentioned signaling pathways. This notion is supported by the finding that phospholipase C (PLC-1PLCE1), a GPCR associated enzyme, is among the non-mitochondrial enhancers, as well as srh-40 (serpentine receptor class H), which is predicted to encode a GPCR. Taken together, we identified many genes among the “non-mitochondrial” enhancers, which regulate intra- and intercellular signaling cascades, and we speculate that these may play a role in signaling of UPRmt, both in a cell autonomous and cell non-autonomous fashion. In addition, we identified “non-mitochondrial” enhancers that directly regulate metabolic homeostasis and, thus, enhance UPRmt in fzo-1(tm1133) mutants.Among the 10 “non-mitochondrial” enhancers of UPRo the ER . Moreovemt are candidates, such as TFG-1TFG and GBF-1GBF1, that encode proteins that have been shown to associate with mitochondria but also other organelles. GBF-1GBF1 is a guanine nucleotide exchange factor (GEF) for the small GTPase ARF-1.2ARF1, which in yeast recruits ARF-1.2ARF1,3 to ER-mitochondria contact sites  and arf-1.2(RNAi). Therefore, the suppression of UPRmt observed in fzo-1(tm1133) animals upon gbf-1(RNAi) may not be due to a rescue of the mitochondrial morphology defect but rather be the consequence of changes in ER-mitochondria contact sites. This highlights the importance of organellar contact sites for the maintenance of mitochondrial and consequently cellular homeostasis. Furthermore, we identified TFG-1TFG, a component of the secretory pathway via COPII vesicles (fzo-1(tm1133)-induced UPRmt. COPII vesicles transport newly synthesized proteins and lipids from specialized ER zones, so-called ER exit sites (ERES), to the Golgi apparatus (copd-1(RNAi) (see above), we speculate that disruption of the secretory pathway may lead to alterations in cellular signaling, ER-mitochondria contact sites and, depending on the context, either to suppression or enhancement of UPRmt. Taken together, we demonstrate that the perturbation of primarily mitochondrial processes leads to the enhancement of UPRmt. However, the identification of non-mitochondrial enhancers demonstrates that disruption of processes taking place outside of mitochondria can also compromise mitochondrial function and activate or enhance UPRmt. Alterations in cellular signaling pathways and/or organellar contact sites may play a role in this respect. Moreover, we find that the majority of suppressors of fzo-1(tm1133)-induced UPRmt are non-mitochondrial, suggesting that many cellular pathways outside of mitochondria exist that can compensate for mitochondrial stress and, hence, ensure mitochondrial homeostasis. In line with this notion, we identified a few “mitochondrial suppressors,” most of which are involved in the maintenance of contacts to other organelles, especially the ER.Among the 16 identified “mitochondrial suppressors” of UPRpparatus . Similarmt, we rescreened the candidates in the background of spg-7(ad2249), which induces UPRmt . spg-7AFG3L2 encodes a mitochondrial matrix AAA-protease, which induces UPRmt when depleted and which is commonly used as a positive control for UPRmt activation  background, in which IMM fusion is blocked. Finally, to identify genes that may modulate UPRmt induced by defects in mitochondrial dynamics, we rescreened all candidates in the drp-1(tm1108) background, in which mitochondrial fission is blocked. In the drp-1(tm1108) background, of the 385 candidates, 291 suppress and 59 enhance. In the eat-3(ad426) background, 242 suppress and 49 enhance. Finally, in the spg-7(ad2249) background, 181 suppress and 54 enhance (Supplementary Table S1).  Since more suppressors reproduced in drp-1(tm1108) and eat-3(ad426) compared to spg-7(ad2249), we conclude that defects in mitochondrial dynamics, to some extent, are suppressed or enhanced by the same pathways. Moreover, the suppressors of fzo-1(tm1133)-induced UPRmt that were sorted into the functional groups “ribosome biogenesis,” “RNA processing” and “translation,” reproduced comparably well in all secondary screens. Thus, attenuation of cytosolic translation may either be a general mechanism to suppress UPRmt or, as discussed above, interfere with reporter expression. Among the enhancers, genes that sorted into the functional groups “ETC assembly factors,” “mitochondrial ribosome biogenesis” and “mitochondrial translation” showed the highest overlap among the secondary screens (Supplementary Table S1), which demonstrates that disruption of mitochondrial translation robustly enhances UPRmt, independent of genetic background.In order to define the specificity of the 299 suppressors and 86 enhancers, we carried out secondary screens. To identify general modifiers of UPRtivation . To idenfzo-1(tm1133) background, we found six enhancers  that did not reproduce in any of the secondary screens. Among these, metl-17METTL17, a methyltransferase required for mitochondrial ribosome assembly and mitochondrial translation in mice -induced UPRmt.While we did not identify any suppressors that act exclusively in the mt in the primary screen using fzo-1(tm1133), enhanced UPRmt in one or more of the secondary screens. Conversely, 10 enhancers of fzo-1(tm1133)-induced UPRmt suppress UPRmt in at least one of the mutants in the secondary screens (listed in the “Opposing UPRmt phenotypes” sheet in Supplementary Table S1). For example, knock-down of icd-1βNAC suppresses Phsp-6mtHSP70gfp in all mitochondrial dynamics-related backgrounds, but enhances spg-7(ad22449)-induced UPRmt. Knock-down of icd-1βNAC in C. elegans has been reported to induce UPRER in wild-type embryos  and drp-1(tm1108) mutants (eat-3(ad426) mutants. Conversely, we speculate that defects in mitochondrial homeostasis induced by a point mutation in spg-7, may exert stress to the cytosol and that this is normally compensated for by factors, such as icd-1βNAC. Knocking-down icd-1βNAC may therefore increase cytosolic stress, which in turn enhances UPRmt in spg-7(ad2249) mutants. Taking the candidates into account that have opposing UPRmt phenotypes in the secondary screens, 95% of the suppressors and 66% of the enhancers reproduce in drp-1(tm1108), while 79% of the suppressors and 57% of the enhancers reproduce in eat-3(ad426). We found the lowest overlap of candidate genes in spg-7(ad2249) mutants, with 59% of the suppressors and 60% of the enhancers reproducing in this background. Taken together, the results of the secondary screens show that there are candidates that, when depleted, act to influence UPRmt signaling in general whereas others are specific to a certain type of UPRmt induction, such as the disruption of mitochondrial dynamics.Twelve candidates that suppressed UPRGATA3,4 and HLH-11TFAP4, which have been shown to play a role in development, aging, and the response to oxidative stress  were identified to be “enhancer-specific”  mutants have previously been shown to be slightly sensitive to oxidative stress and have increased levels of carbonylated proteins, a measure for oxidative damage (isp-1(qm150) and clk-1(qm30) mutants, both of which have increased levels of reactive oxygen species (ROS)  mutants most likely through increased metabolic activity (mt in fzo-1(tm1133) mutants acts to compensate for metabolic defects. In summary, we identified several TFs with roles in development, oxidative stress response and metabolism that previously have not been connected to UPRmt signaling. These TFs may be specific to UPRmt in fzo-1(tm1133) but some may generally be involved in UPRmt signaling.Three TFs . Next, we manually annotated the C. elegans orthologs of 24 interactors of Mfn1 or Mfn2 in mammals  but again did not find any overlap between the gene network and our screen dataset . In summary, in our screen for modifiers of fzo-1(tm1133)-induced UPRmt, we did not find any previously known interactors of fzo-1MFN1,2. These could either have been missed in the RNAi screen, be essential in the fzo-1(tm1133) background or not have a function in mitochondrial homeostasis and, hence, UPRmt signaling.In order to determine whether any of the suppressors or enhancers that we identified have previously been shown to interact with C. elegans genes currently associated with the GO-term “mitochondrial unfolded protein response” (GO: 0034514) (referred to as “input genes”), identified their human orthologs and included known physical and genetic interactors from the interaction databases “BioGRID3.5,” “IntAct,” and “mentha” , 36 of which are enhancers and 77 of which are suppressors of fzo-1(tm1133)-induced UPRmt, with a total of 213 interactions  that suppress fzo-1(tm1133)-induced UPRmt and the gene products of four of these localize to the nucleus   has previously been reported to not cause splicing defects -induced UPRmt upon knock-down. taf-4TAF4 has previously been shown to be required for life span extension in isp-1(qm150), clk-1(qm30) and tpk-1(qm162) mutants, , which enhances fzo-1(tm1133)-induced UPRmt and plays a central role in the inositol triphosphate (IP3) signaling pathway  signaling   to the plasma membrane P2 to generate the second messengers DAG and IP3, known regulators of several signal transduction pathways , thereby affecting mitochondrial homeostasis and consequently UPRmt signaling. Moreover, we propose that the effect on UPRmt signaling may be indirect since we previously showed that knock-down of mitochondrial genes controlling calcium homeostasis does not induce UPRmt in wild type (fzo-(tm1133) mutants may be more prone to changes in IP3 signaling and, consequently, calcium signaling since these mutants may have altered ER-mitochondria contact sites, as shown in tissue culture cells lacking the mammalian ortholog MFN2  , and EFRP5K1A  were found to regulate mitochondrial fusion and to be critical for mitochondrial function in human tissue culture cells and in mice (K01D12.6 “mitoguardin homolog-1 (miga-1).” We verified UPRmt induction using the Phsp-60HSPD1gfp (zcIs9) reporter in the miga-1(tm3621) mutant background (hsp-60HSPD1gfp is higher in miga-1(tm3621) animals compared to fzo-1(tm1133) animals. Moreover, we tested the effects of miga-1(tm3621) on steady-state mitochondrial morphology, which, in C. elegans, is carried out using a mitochondrial matrix-targeted GFP under a promoter that expresses the transgene in body wall muscle cells (Pmyo-3MYHmtgfp) (miga-1(tm3621) mutants have a “fragmented mitochondria” phenotype, which is less severe than that caused by the loss of fzo-1 (miga-1(tm3621) mutants. Specifically, for most of the shape descriptors analyzed, miga-1(tm3621) mutants were statistically different from wild type but distinct from fzo-1(tm1133) mutants (miga-1(tm3621) mutants, which are accompanied by the induction of UPRmt.One of the enhancers we identified is ckground . On averof fzo-1 . In addi mutants . In summ


Targeting the KRAS pathway is a promising but challenging approach for colorectal cancer therapy. Despite showing potent efficacy in BRAF-mutated melanoma, MEK inhibitors appeared to be tolerated by colorectal cancer cells due to their intrinsic compensatory signaling. Here, we performed genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitor to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. Several genes were identified as potential functional drivers, which were significantly enriched in the GRB7-mediated RTK pathway. Loss-of-function and gain-of-function assays validated that GRB7 potently rendered CRC cells primary resistance to MEK inhibitors through the RTK pathway. Mass spectrum analysis of GRB7 immunoprecipitates revealed that PLK1 was the predominant interacting kinase of GRB7. Inhibition of PLK1 suppressed downstream signaling of RTK, including FAK, STAT3, AKT, and 4EBP1. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. In conclusion, we identified GRB7-PLK1 as a pivotal axis mediating RTKs, resulting in MEK inhibitor tolerance. PLK1 is therefore a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients harboring KRAS mutations. Colorectal cancer (CRC) is one of the most common malignancies, with the third highest incidence and death rate in both women and men in the United States . ApproxiSince RAS can activate the MAPK pathway, which consists of a linear kinase cascade, blockade of downstream kinases, vertical repression of MEK, has previously been considered a promising strategy . HoweverMultiple high-throughput genome-wide screening approaches have been applied to unbiasedly identify critical genes that contribute to drug resistance in human cancers. Previously, RNA interference (RNAi) screening with shRNA library to knockdown targeted genes has been widely used , 19. HowTherefore, we performed genome-wide CRISPR/Cas9 knockout screening to identify the pivotal oncogenes contributing to overcoming MEKi resistance in CRC cells. Further, we performed co-targeting of MEK and PLK1 kinases to investigate its effect on CRC both in vitro and in vivo. Our findings will not only help clarify the mechanism of primary resistance to MEK-targeted therapy in CRC, but also provide a combinatorial regimen for clinical treatment.To identify the critical driver genes involved in MEKi resistance, we designed a combination strategy with genome-wide CRISPR/Cas9 knockout library screening and transcriptomic analysis of KRAS-mutated cell lines Fig. . The modAmong the genes involved in the RTK pathway, growth factor receptor-bound protein 7 (GRB7) was identified as the most negatively selected gene after AZD6244 treatment Fig. . Five ouTo validate the function of GRB7 in terms of driving primary resistance to MAPKi using an orthogonal approach, we evaluated CRC cell proliferation in the presence of MEKi with shRNA-mediated perturbation of GRB7. The efficiencies of shRNAs were determined by qPCR, and optimized sequences were used in the functional experiments . Potential GRB7-interacting proteins were co-precipitated with anti-FLAG antibody-conjugated beads and subsequently subjected to MS analysis. A total of 57 GRB7-interacting proteins were enriched at least threefold in HCT116 cells expressing FLAG-GRB7 compared to that in control cells  model. HCT116 and SW480 cells were cultured in Matrigel and treated with DMSO, MEKi, PLK1i, or their combination for 8 days. One representative sphere was tracked continually and photographed at the indicated time points. Although MEKi or BI-2536 treatment partially suppressed CRC spheroid growth, BI-2536 treatment significantly improved the therapeutic effects of MEKis. The combination regimen not only significantly inhibited cell proliferation in spheroid numbers and sizes, but also induced apoptosis in HCT116 and SW480 cells Fig. . EncouraFurthermore, similar to the inhibition of PLK1, knockdown of RPTOR also sensitized HCT116 and SW480 cells to AZD6244  and 1% penicillin/streptomycin. All cell lines were purchased from ATCC and verified to be free of mycoplasma.HT29, WIDR, HCT116, LS174T, and HEK293T; SW480 and A549; and CFPAC-1 cells were cultured in DMEM; RPMI; and IMDM, respectively, in a humidified 5% CO+/PI−) and late apoptotic cell population (Annexin V+/PI+) were combined to obtain the total proportion of apoptotic cells.Cell apoptosis assays were performed as previously described . Cells wSupplementary informationSupplementary Table S1Supplementary Table S2Supplementary Table S3


Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte’s physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca Cardiovascular medicine is presently facing a new challenge with rapidly rising numbers of patients suffering from heart disease, the leading cause of death worldwide . Because2+ channels (LTCCs) and the ryanodine receptors (RyR2s), the Ca2+ release channels of the SR. Both channels are fundamental to the mechanism of excitation-contraction (EC) coupling and control cytosolic Ca2+ entry by Ca2+-induced Ca2+ release (CICR), which links electrical excitation to contractile activity and force production by the myocyte in a highly spatiotemporally synchronized way  to the silicone-based organic polymer polydimethylsiloxane (PDMS) of different softness . We focused on intracellular Ca® medium  at a density of 2×104 cells per dish for Ca2+ measurements and on glass coverslips in 24-well plates  for immunostainings, and at 104 cells per dish onto glass-bottom dishes  and PDMS-coated dishes  for electrophysiological experiments and live-cell imaging analysis.Murine iPSC-CMs were received from Ncardia  and kept in liquid nitrogen until use. Culture dishes were coated with a mixture of laminin and fibronectin in PBS (1:1:100) overnight at 37°C to enable attachment of iPSC-CMs. After defrosting, cells were seeded in Cor.AT® medium. Cells were maintained in culture at 37°C and 5% CO2 and used within 4weeks.To avoid the growth of undifferentiated cells and non-cardiomyocytes, the cardiomyocyte-specific α-myosin heavy chain (α-MHC) promoter was chosen to control pac gene expression for puromycin resistance. Puromycin (1μg/ml) was added for the selection of cardiac-specific cells for the first 48h in culture. Afterward, cells were kept in culture in puromycin-free Cor.At2 1.8, MgCl2 1.1, HEPES 5, glucose 10, pH 7.4.For all live-cell imaging experiments, the standard bath solution was cardiac Tyrode’s solution containing (in mm): NaCl 140, KCl 5.4, CaCl2+ transients of iPSC-CMs were recorded using the ratiometric Ca2+-sensitive fluorescent indicator fura-2AM . Cells were loaded with 1.5μm fura-2AM diluted in Tyrode’s solution and incubated for 20min, followed by 10min of de-esterification. iPSC-CMs were constantly perfused with prewarmed Tyrode’s solution containing Probenecid (100μm) to avoid sequestration or secretion of fura-2. Using the IonOptix system , Ca2+ transients were recorded in parallel with edge detection to measure contractions. Data were collected by using the IonWizard software developed by IonOptix. Cells were exposed to light emitted by a xenon lamp passing through rapidly switching filters of 340nm and 360nm to determine the ratio of bound and unbound Ca2+ ions in the cells. Fluorescence emission light was collected at 510nm. Data are presented as fura-2 ratio (F340/F360).Ca2+ transients and contractions at steady state were analyzed per cell using OriginPro® software . Assessed parameters comprised peak Ca2+ transients and shortening amplitudes, time-to-peak (TTP), full duration at half maximum (FDHM), decay, and frequency of spontaneous activity. Decay of contractions was fitted with a Boltzmann function, whereas decay of Ca2+ transients was calculated by an exponential decay function. Diastolic Ca2+ levels of paced iPSC-CMs were only evaluated if cells responded to the frequency of electrical stimulation at 1Hz and 2Hz (10V) by a field stimulator .For functional evaluation of spontaneous activity, only rhythmically beating iPSC-CMs were used. Five representative Ca2+ currents (ICaL) were measured in iPSC-CMs via the patch-clamp technique with simultaneous recording of Ca2+ signals by confocal line-scan imaging. Experiments were performed using a HEKA EPC-10 patch-clamp amplifier  connected to an Olympus IX81 laser scanning confocal microscope . For patch-clamp recordings, borosilicate glass pipettes were pulled to obtain tip resistances of 2–9 MΩ and filled with an internal solution containing 8mm NaCl, 120mm CsAsp, 20mm TEA-Cl, 5.9mm MgCl2, 20mm HEPES, 5mmK2-ATP, and 50μm of the Ca2+-sensitive fluorescent indicator K5-fluo-3 . For ICaL measurements, iPSC-CMs were constantly perfused with warm Tyrode’s solution (37°C) containing 5mm CsCl.For investigation of the EC-coupling mechanism, Ca+ channels by a 500ms ramp ranging from the holding potential of −80mv to −40mv, the first test step was set to −25mv for 400ms, followed by a second 400ms test step from −40mv to +10mv to record maximal ICaL and Ca2+ release amplitudes.For analysis of the EC-coupling gain, a two-step protocol was applied  and/or cells with low fluo-3 loading (F/F0<1.2) were excluded from analysis. As a control, the ratio of the maximal Ca2+ transient and current amplitudes at +10mv was calculated. ICaL at +10mv was used for the measurement of maximal current amplitude and inactivation kinetics. Fitting ICaL with a biexponential function in OriginPro allowed the comparison of tau1 (τ1) as an indicator of Ca2+-dependent inactivation.For analysis of the EC-coupling gain, the ratio of the peak Ca2+ content, caffeine-elicited NCX currents were recorded.For examination of fractional release and NCX activity in iPSC-CMs, patched cells were stimulated to steady-state activity at a frequency of 1Hz, followed by application of the RyR2 agonist caffeine  for total depletion of the SR. In parallel with the measurement of SR CaFor functional evaluation of gap junctions, fluorescence recovery after photobleaching (FRAP) was measured using the gap junction-permeant dye calcein . Photobleaching and imaging were done on the Olympus FluoView LSCM using a 60x water immersion objective (1.2 NA). iPSC-CMs were loaded with 0.5μm calcein-AM in Tyrode’s solution for 20min. After 10min of de-esterification in Tyrode’s solution, calcein-diffusion dynamics between iPSC-CMs were assessed as an indicator of functional Cx43 expression and gap junction formation. One cell in a cluster was bleached with a laser power of 50% at 10μs/pixel for 5s, and fluorescence recovery was recorded in 52 images taken every 10s with a laser power of 0.5–1.5% at 2μs/pixel. Importantly, the target cell had to be fully surrounded by neighboring cells (in 2D), thus representing one building block in a conductive and connected cell layer. Further analysis was done in ImageJ to plot the time course of fluorescence recovery of the bleached cell. After subtraction of background and bleaching point, the graph was normalized to the initial value of fluorescence intensity (before bleaching). The final traces were fitted in OriginPro software with a biexponential function to investigate the fast time constant 1 (τ1) as an indicator of diffusion rate.2+ handling proteins. Cells grown on PDMS-coated dishes were fixed at days 10, 20, and 25 after seeding, cells on glass coverslips after 9, 13, and 20days. To block unspecific binding sites, cells were incubated with bovine serum albumin dissolved in PBS  for 0.5h at room temperature. For permeabilization of iPSC-CMs, the blocking solution contained 0.1% Triton X-100. Cells were incubated with primary antibodies against Cx43 , SERCA , NCX1  or RyR2 , and Cav1.2  at room temperature for 90min. After three times washing with PBS for 10min, cells were incubated with appropriate secondary antibodies conjugated to Alexa Fluor dyes  with different excitation-emission spectra for 1h. After three times washing with PBS for 10min, samples were incubated for 1h in Phalloidin-TRITC  for actin staining. After final washing steps in PBS, cells were mounted with fluoroshield containing DAPI for nuclei staining .For immunostainings, cells were washed with PBS, fixed with 4% paraformaldehyde  for 20min, and washed three times with PBS for 10min. Different time points were chosen to examine the influence of growth duration on the expression pattern and spatial organization of different Cav1.2 expression. Laser excitation at 405nm, 488nm, 552nm, and 638nm was used in sequential scans. Emission spectra were chosen via tunable filters, and emission detection was achieved by photomultiplier tubes (PMT) and hybrid detectors (HyD). Image processing and Cx43 sarcolemmal expression analysis were done using ImageJ/Fiji as described in  using the acquisition software LAS-X  for high-resolution images. 20x oil immersion objective was selected to record an overview of Cx43 stainings, and 63x oil immersion objective was used for detailed imaging of SERCA, NCX1, RyR2, and Caribed in . Due to ® software  was used. Images were processed in ImageJ. Data are presented as mean±standard error of the mean (SEM) with n equaling the number of individually analyzed cells. Experiments were repeated in five rounds with iPSC-CMs defrosted from five individual vials at different time points . Depending on the data set, statistical significance was determined by Student’s t-tests or one-way ANOVA followed by multiple comparison tests, indicated by * for p<0.05.For statistical data analysis and graph design, OriginProFor live-imaging and functional analysis, cells are usually seeded on rigid glass surfaces, which present an unphysiologically high stiffness of 25GPa. In order to investigate the impact of different physiological substrate stiffnesses on the maturation potential of iPSC-CMs, cells were seeded at high density on glass and for comparison on PDMS culture surfaces with stiffnesses of 28kPa, 15kPa, and 1.5kPa, respectively, corresponding to the environment of neonatal and adult cardiomyocytes. All surfaces were coated with laminin and fibronectin for cell adhesion. Different growth materials did not have any impact on cell adhesion or cell survival during the culture time of up to 4weeks (data not shown). But a simple examination of cell contractile behavior revealed that compared to glass, the contractile activity of iPSC-CMs appeared stronger and temporally better synchronized across the cell monolayer  indicati2+ handling and contractile activity in iPSC-CMs, spontaneous Ca2+ transients and contraction dynamics were measured in cells seeded on PDMS-coated or glass-bottom dishes. Cells were loaded with the fluorescent Ca2+-sensitive dye fura-2AM (1.5μm) to record Ca2+ transients, and cell shortening was optically measured via edge detection. Due to their immaturity, iPSC-CMs showed high variability in their spontaneous activity pattern. For evaluation of comparable cells, we categorized iPSC-CMs into three different groups of rhythmic, arrhythmic, and oscillating cells  using the whole-cell patch-clamp technique and recorded simultaneously intracellular Ca2+ transients by confocal imaging in the line-scan mode. As demonstrated in via patch-clamp, a two-step protocol was applied to measure ICaL at negative potentials and at maximal current activation. In parallel, changes in cytosolic Ca2+ levels were recorded by confocal imaging of fluo-3 included in the patch pipette solution. Starting first from a holding potential of −80mv, a 500ms voltage-ramp to −40mv was applied to activate and immediately inactivate the fast voltage-dependent Na+ current. After 800ms at −40mv, the first test step was applied. Em was set to −25mv to activate ICaL and CICR at low amplitude. The second test step to +10mv fully activated ICaL and served as control measurement for ICaL and CICR. Changes in fluorescence intensity were plotted over time to generate line profiles from the line-scan image, as depicted in ICaL and Ca2+ transients at +10mv showed similar amplitudes in iPSC-CMs grown on substrates with different stiffnesses . The EC-coupling gain at −25mv was significantly increased in comparison with +10mv when stiffness was 15kPa (p=0.0445) or 1.5kPa  in iPSC-CMs grown on surfaces with different stiffness. 2+ transients were recorded simultaneously. Peak amplitudes of steady-state Ca2+ transients and caffeine-induced Ca2+ transients are summarized in 2+ load , relative shortening amplitude was significantly larger in iPSC-CMs grown on 1.5kPa-PDMS compared to higher stiffnesses of glass, 28kPa-PDMS or 15kPa-PDMS, indicating significantly enhanced contractility  on substrates with different stiffnesses revealed significantly higher myofilament Ca2+ sensitivity in iPSC-CMs seeded on 1.5kPa-PDMS and 15kPa-PDMS compared to glass . These results demonstrate significantly enhanced diffusion dynamics of calcein and therefore an increased presence of functional gap junctions, which allowed faster FRAP in iPSC-CMs grown on soft surfaces.For functional evaluation of gap junctions, we analyzed FRAP using the gap junction permeant fluorescent dye calcein-AM (0.5mM). After removal of the acetoxy-methylester group of calcein by cytosolic esterases, the dye was no longer able to diffuse across the lipid bilayer. Consequently, gap junctions presented the only possibility for calcein to leave intact cells. Calcein was bleached in one cell of a cell group, and fluorescence recovery of calcein diffusing in from neighboring cells was measured over time revealing the diffusion dynamics between iPSC-CMs . RepreseFor the development of an adult heart with its unique electrophysiological and contractile properties, numerous specific growth conditions are required. Electrical stimulation by pacemaker cells, humoral regulation, and the balance between extrinsic and intrinsic mechanical load are just some of the essential factors that influence structural and functional maturation of cardiomyocytes during the early stages of development . In thisin vitro. The goal of this study was therefore to examine the impact of the environmental stiffness on cardiomyocyte function at the level of EC-coupling and intercellular communication in iPSC-CMs and to find out whether an environment with physiological stiffness may provide a new possibility to enhance mature properties of iPSC-CMs.Considering cardiomyocytes derived from pluripotent stem cells, investigations of MCT may reveal new targets for improving their functional properties to enhance their potential to be used for cardiac cell therapy. In other words, a detailed analysis of the influence of specific environmental cues on elementary features of iPSC-CMs may help to mature these cells 2+ handling in response to a specific treatment. iPSC-CMs plated on soft surfaces of 15 or 1.5kPa-PDMS showed greater Ca2+ release upon triggering influx current at −25mv, indicative of better coupling between LTCCs and RyR2s. Considering possible explanations for this enhanced EC-coupling gain, changes in the expression pattern of LTCCs and RyR2s, i.e., a switch from punctuate to regular striation pattern, may explain this finding very well and indicate structural remodeling toward maturation. In this new arrangement, the number of non-coupled RyR2s is reduced, while the increased colocalization of LTCCs and RyR2s favors higher EC-coupling efficiency in iPSC-CMs. Another possibility for the enhanced EC-coupling gain might be an increase in the Ca2+ sensitivity of RyR2s due to posttranslational modifications. In addition to reactive oxygen and nitrogen species, Ca2+-dependent RyR2 activation is physiologically regulated by phosphorylation via protein kinase A (PKA) and Ca2+/calmodulin-dependent kinase II , more Ca2+ is being released from the SR and therefore available for myofilament activation. In addition, the contractile response to different surfaces in adult cardiomyocytes may also depend on adaptions in myofilaments, an idea that also seems to apply to iPSC-CMs  and iPSC-CMs caused by weak intercellular coupling poses a high risk for the development of a conduction barrier and arising arrhythmias . In our 2+ handling properties leading to enhanced EC-coupling gain and contractility in iPSC-CMs. Moreover, this study provides the first experimental evidence of an increased presence of functional gap junctions, which lead to better intercellular coupling with stronger synchronization of the electro-mechanical activity in iPSC-CMs grown on soft surfaces. By exhibiting relevant targets of MCT in iPSC-CMs, our results may trigger further investigation of mechanical signaling pathways leading not only to new maturation strategies of iPSC-CMs, but also to the identification of new targets for the treatment of cardiac diseases with changed ECM stiffness.In conclusion, here we showed non-linear cellular responses to substrates with different stiffnesses at a macroscale level in iPSC-CMs indicating the existence of MCT. We demonstrated that soft growth surfaces trigger structural maturation at the level of CICR and promote CaThe original contributions presented in the study are included in the article/NU conceived the study. AK and NU designed the experiments, conducted the experiments, analyzed and interpreted the data, generated the figures and wrote the manuscript. MM and MH critically reviewed and edited the manuscript. All authors have reviewed the data and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The delivery of cancer therapeutics can be limited by pharmacological issues such as poor bioavailability and high toxicity to healthy tissue. pH-low insertion peptides (pHLIPs) represent a promising tool to overcome these limitations. pHLIPs allow for the selective delivery of agents to tumors on the basis of pH, taking advantage of the acidity of the hypoxic tumor microenvironment. This review article highlights the various applications in which pHLIPs have been utilized for targeting and treating diseases in hypoxic environments, including delivery of small molecule inhibitors, toxins, nucleic acid analogs, fluorescent dyes, and nanoparticles. Toxicity to healthy tissue can limit the dose and timing of several therapeutics especially cancer therapeutics, impacting quality of life and causing delays in treatments. Therefore, the development of tumor-specific agents would represent an important step towards overcoming these limitations. To date, much of the effort to develop targeted agents with relative tumor specificity has relied on the surface expression of tumor-specific antigens or cancer-specific dependence on signaling pathways. While these approaches have all led to important and meaningful improvements in cancer therapy, by nature of their design they are limited to specific cancer types and subtypes.pH-low insertion peptides (pHLIPs) represent a potential system to specifically target tumors, and importantly do not rely on the surface expression of tumor-specific antigens. Instead, pHLIPs are a pH-based delivery system that take advantage of the acidic condition of the tumor microenvironment for selective delivery of cargo. Acidity of tumor environments arise mainly as a consequence of hypoxia. As acidity is a ubiquitous hallmark of the tumor microenvironment, pHLIPs have potential for use across a number of different tumor types. This review article outlines pHLIP function and describes the numerous ways that pHLIP has been used in experimental models to demonstrate efficacy for cancer therapy and/or imaging.In normal tissue, extracellular pH is tightly regulated to approximately 7.4, while in pathological states, such as ischemia, inflammation, and neoplasia, dysregulation of pH commonly occurs. The tumor microenvironment is largely acidic , likely pH-low insertion peptides (pHLIPs) demonstrate pH-dependent ability to insert across cell membranes  and are Cargo can be linked to the C-terminus of pHLIP using a disulfide bond or other linkers that are subject to cleavage in the intracellular reducing environment . LinkageImportantly, the mechanism of action of pHLIP confers three advantageous properties for potential cancer therapeutics. First, the pH-specificity of pHLIP confers relative selectivity in targeting tumor cells, which allows for sparing of normal tissue and therefore an expected reduction in dose-limiting toxicities. Furthermore, the pHLIP system can assist in the transport of otherwise cell-impermeable molecules across the cell membrane, ameliorating potential issues in bioavailability. Finally, pHLIP targets the tumor microenvironment on the basis of acidity, a ubiquitous feature of the tumor microenvironment, thereby distinguishing it from other attempts to develop tumor-specific reagents.In mouse models, fluorescently labeled pHLIP has shown targeting to subcutaneously injected and spontaneous tumors, as well as metastatic lesions  6]. Acc. Acc6]. pHLIP shows enhanced targeting to aggressive tumors, as evidenced by the enhanced uptake of fluorescently labeled pHLIP by tumors formed from M4A4 cells, a highly aggressive and metastatic melanoma line, as compared with tumors formed from NM2C5 cells, a less acidic and weakly metastatic melanoma cell line . The abiMany cancer therapeutics are limited by their inability to cross the blood–brain barrier, limiting potential treatment options for brain cancers and metastases. Biodistribution experiments in orthotopic and subcutaneous models of glioma show accumulation of pHLIP is limited to extracranial sites, suggesting that pHLIP is unable to cross an intact blood–brain barrier . HoweverBeyond the tumor microenvironment, a number of other pathological states involve the dysregulation of tissue pH, including inflammation . In addi50 (pH at which 50% of pHLIP are in the inserted state) of pHLIP to improve cell uptake. pHLIP-D25E-C (Asp25 is substituted with Glu), pHLIP-P20G-C (Pro substituted with Gly) have higher pH50, whereas pHLIP-R11Q-C (Arg is substituted with Gln) exhibit lower pH50 AlF-cysVar3 pHLIPAcidity is a characteristic common to tumors that occurs partly due to metabolic alterations in response to hypoxic microenvironments. In animal models, exploiting acidity for targeted tumor delivery has been achieved using pHLIP for therapeutic and imaging purposes. Conjugating molecules to the inserting end allows cytoplasmic delivery, whereas labeling of acidic cells can be achieved by conjugating to the N-terminus. Multiple variants of pHLIP with altered pharmacokinetic properties have been reported that can be manipulated for various applications. pHLIP precisely delivers small polar toxins, DNA repair inhibitors, imaging agents, nanoparticles and antisense peptide nucleic acids to acidic environments in vivo. PNAs stand out among the numerous cargos delivered by pHLIP since they are non-toxic, sequence specific DNA mimics that can regulate gene expression by targeting microRNAs and mRNAs and can be effective against oncologic targets that are otherwise undruggable. The use of pHLIPs to deliver highly potent small molecule toxins to tumors may also provide advantages over antibody-drug conjugates for the same purpose because pHLIP targeting does not depend on expression of a specific antigen but instead exploits the acidity of solid tumors.While pHLIP remains a promising technology, improving the synthetic efficiency of pHLIP cargo constructs could significantly facilitate its clinical translation. In recent years, the discovery of multiple variants of pHLIP and new conjugation approaches have expanded the possibilities of cargoes that pHLIP can deliver to the acidic and hypoxic tumor microenvironment. Ultimately, this raises new potential for the use of pHLIP in the treatment and diagnosis of cancer.


Together, our findings unravel a functional role of DNAM-1 in AML pathophysiology and identify DNAM-1 as a potential novel prognostic maker in AML.DNAM-1 is reportedly expressed on cytotoxic T and NK cells and, upon interaction with its ligands CD112 and CD155, plays an important role in tumor immunosurveillance. It has also been reported to be functionally expressed by myeloid cells, but expression and function on malignant cells of the myeloid lineage have not been studied so far. Here we analyzed expression of DNAM-1 in leukemic cells of acute myeloid leukemia (AML) patients. We found substantial levels of DNAM-1 to be expressed on leukemic blasts in 48 of 62 (> 75%) patients. Interaction of DNAM-1 with its ligands CD112 and CD155 induced release of the immunomodulatory cytokines IL-6, IL-8 IL-10 and TNF-α by AML cells and DNAM-1 expression correlated with a more differentiated phenotype. Multivariate analysis did not show any association of DNAM-1 positivity with established risk factors, but expression was significantly associated with clinical disease course: patients with high DNAM-1 surface levels had significantly longer progression-free and overall survival compared to DNAM-1 Diagnostic classification of AML is based on morphologic, immunophenotypic, cytogenetic and molecular genetic analyses3. Rapid and accurate risk stratification is essential for guiding treatment decisions and predicting therapy outcomes, and novel prognostic markers may thus improve clinical management of patients. So far, immunophenotyping of AML blasts plays a great role in diagnosis but is less important for risk assessment. Besides typical myeloid surface markers, abnormal expression of various markers of non-myeloid lineages on AML blasts has been reported5.Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults6. Besides the well documented role of DNAM-1 in lymphocytes, expression was also reported for cells of the myeloid lineage like monocytes and platelets, where it mediates adhesion and migration8 However, nothing is known regarding the expression and function of DNAM-1 on malignant cells of the myeloid lineage. Notably, aberrant expression of immune receptors normally found on NK cells was previously reported for AML cells, with the Fcɣ receptors CD32, CD64 and CD16 being prominent examples12.DNAX accessory molecule-1  is a transmembrane nectin-like glycoprotein of the immunoglobulin superfamily and acts as an activating receptor on NK cells and T cells. Its ligands, CD112 and CD155, are likewise members of the nectin family, and the DNAM-1-CD112/CD155 axis is mainly known for its important role in NK cell-mediated killing of tumor cellsIn this study we report that DNAM-1 is frequently expressed on AML patient cells, demonstrate that DNAM-1 stimulates the production of immunomodulatory cytokines by the leukemic cells and identified a significant correlation of DNAM-1 with disease outcome.Peripheral blood mononuclear cells (PBMC) of AML patients were isolated by density gradient centrifugation and frozen in liquid nitrogen. Informed consent was obtained from all patients in accordance with the Helsinki protocol. In this study all experimental protocols were conducted according to the guidelines and were approved by the local ethics committee of the University of Tübingen .DNAM-1 mAb clone 102511 and mouse IgG1 isotype control were from R&D Systems and BD Biosciences, respectively. All fluorescent antibody conjugates  were from BD Biosciences and used in 1:200 dilution, secondary goat-anti–mouse-PE was from Dako. Recombinant human CD112 or CD155 Fc-domain fusion proteins (rhCD112/rhCD155) were from BioLegend. Recombinant proteins were tested to be free of endotoxins by ENDONEXT EndoZyme II assay (bioMérieux).The following cell lines were obtained from German Collection of Microorganisms and Cell Cultures : KG-1, EOL-1, Kasumi-1, K562, HL-60, TF-1 and U937. Authenticity was routinely determined by validating the immunophenotype described by the provider using flow cytometry. Cells were cultured in RPMI1640 medium (Gibco) supplemented with 10% of heat-inactivated fetal bovine serum (PANBiotech) and 1% penicillin–streptomycin (Lonza).DNAM-1 surface expression was assessed by staining with a specific antibody or isotype control followed by incubation with goat-anti–mouse IgG-PE. Samples were analyzed using FACSCanto II instrument (BD Biosciences).Leukemic cells in patient samples were first selected by FSC/SSC and then gates were applied according to the surface markers based on the individual immunophenotype defined upon routine diagnosis . Dead cells were excluded based on 7-aminoactinomycin staining . Specific fluorescence indices (SFI) were calculated by dividing median fluorescence values measured with specific antibody by median fluorescence values obtained with isotype control.6 AML cells using High Pure RNA kit (Roche). cDNA was synthesized from 500 to 1000 ng RNA using Go Script Reverse Transcription System (Promega) according to the manufacturer's instructions. DNAM-1 cDNA was amplified using 2 × qPCR SyGreen Mix (PCR Biosystems) using primers 5′-GTGGAGTGGTTCAAGATCGGG-3′ and 5′-GCTTCCTTATGACCATGCCAT-3′ (79 bp). Reference gene (GAPDH) was amplified using primers 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′ (225 bp). PCR was performed using LightCylcer-480 instrument (Roche). Amplicons were additionally analyzed by gel electrophoresis.Total RNA was isolated from 1 × 106 cells/ml. Cytokines in supernatants from cultured AML blasts were quantified by ELISA according to the manufacturer's instructions using OptEIA assays from BD Pharmingen. Viability was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions.For stimulation with DNAM-1 ligands, 96-well cell culture plates (Greiner Bio-One) were coated with 10 µg/ml solution of rhCD112, rhCD155 or hIgG1 in PBS overnight. Afterwards wells were washed with PBS and freshly thawed AML cells were plated in concentration 2 × 10+ versus CD34- cells from the same patient paired Wilcoxon rank test was used. Survival curves were analyzed by the Kaplan–Meier method. Log-rank test was performed for estimating survival differences between the groups. Cutoff value for separation of individuals into DNAM-1high and DNAM-1low was determined using receiver operating characteristic (ROC) analysis in JMP® Pro as previously described 13. Value of highest Youden’s index was used as cutoff. P values of < 0.05 were considered statistically significant. Patients with missing data were excluded from statistical analysis.Statistical analysis was conducted using GraphPad Prism 8.1.0 and JMP® Pro  software. The two-tailed unpaired Mann–Whitney or Kruskal–Wallis tests were used to compare individual groups. For comparison of DNAM-1 expression in CD34The clinical characteristics of the patient cohort are summarized in Table As a first step, we used flow cytometry to determine DNAM-1 surface expression on cancer cell lines representing a wide range of myeloid malignancies: K562 (chronic myeloid leukemia), U937 (pro-myelocytic leukemia), KG-1 (AML M6), EOL-1 (AML M4), Kasumi-1 (AML M2), HL-60 (AML M2), TF-1 (AML M6) Fig. . NotablyNext, we analyzed DNAM-1 expression on leukemic blasts from AML patients. The gaiting strategy and examples of surface staining are shown in Fig. 16, we next set out to determine whether DNAM-1 was functional in AML cells. To this end we analyzed cytokine production in cultures of primary AML specimens upon stimulation with the two different DNAM-1 ligands CD112 and CD155. We selected thirteen AML samples with high DNAM-1 expression on leukemic cells  and incubated them on immobilized recombinant human DNAM-1 ligands (rhCD112-Fc or rhCD155-Fc) or isotype control (human IgG1) for 24 h. Afterwards secretion of the immunomodulatory cytokines IL-6, IL-8, TNF-α and IL-10 that are implicated in AML pathophysiology18 were analyzed by ELISA. Exemplary cytokine profiles obtained with 2 DNAM-1-positive (UPN34 and UPN20) and 1 negative specimen (UPN18) as control are shown in Fig. 19, induced release of cytokines in DNAM-1 negative samples, confirming that effects observed with positive leukemic cells were specifically mediated by DNAM-1 signaling . First, we correlated the intensity of DNAM-1 surface staining with morphological characteristics according to the FAB classification. The fractions of positive (SFI ≥ 1.5) samples among analyzed patients with different FAB types were as follows: M0, 3 of 4 (75%); M1, 6 of 12 (50%); M2, 5 of 9 (56%); M3, 6 of 7 (86%); M4, 11 of 12 (92%); M5, 4 of 5 (80%) and M6, 10 of 10 (100%). The smallest mean DNAM-1 positivity (SFI > 1.5) was found among M1 subtype samples (6.7%), the highest positivity was observed with FAB M5 samples (mean positive cell fraction 51%)  and assayed DNAM-1 expression on CD34+ versus CD34- cells. We found that DNAM-1 positive cells were enriched in the CD34- population  and DNAM-1low (n = 39) groups   in each quartile was assessed. Kaplan–Meier analysis did not reveal any difference in survival between the four groups p = 0.49) 22. Whether DNAM-1 is expressed in malignant myeloid cells and whether it contributes to disease pathophysiology is so far unknown.DNAM-1 is an important mediator of effector functions in NK cells and T cells. Besides cytotoxic lymphocytes, functionally relevant DNAM-1 expression was described on B cells, but also on healthy cells of the myeloid lineage like monocytes and platelets23. No correlation between the extent of cytokine release and the surface levels of DNAM-1 was observed, suggesting that the signaling capacity of the receptor is influenced by other factors, for example by co-expression of inhibitory molecules such as CD9625.Here we report that DNAM-1 is expressed in cell lines of myeloid lineage and primary leukemic cells of AML patients. When we investigated the functionality of the DNAM-1 in AML cells, we found that interaction with its ligands resulted in production of the cytokines IL-6, IL-8, TNF-α and IL-10. This is in line with reports that interaction of DNAM-1 on cytotoxic cells with CD155 or CD112 triggers signaling via immunoreceptor tail tyrosine (ITT)-like motifs and stimulates cytokine secretion31 and associate with disease outcome34. IL-10 reportedly influences proliferation and cytokine production by AML cells 36. Elevated plasma levels of IL-10 correlate with better survival of patients. For TNF-α controversial effects in AML pathogenesis have been reported37. Notably, variable patterns of cytokine release upon DNAM-1 signaling were observed. Whereas IL-6 or IL-8 were released by more than a half of the samples tested, secretion of IL-10 and TNF-α was observed less frequently. Likely the ultimate effect of DNAM-1 signaling may be is determined by combinatorial action of several secreted factors. Regarding the association of DNAM-1 expression and clinical parameters, we found that more mature AML cells displayed higher DNAM-1 positivity compared to immature FAB subclasses. Thus, DNAM-1 may serve as additional maturation marker upon immunophenotyping. Further in line with the association of DNAM-1 positivity and a more differentiated phenotype, we observed substantially higher expression on mature CD34-negative subpopulations of leukemic cells.IL-6 and IL-8 are implicated to affect cellular survival in various cancer entities including AML40, and expression associates with prognosis42. To evaluate whether DNAM-1 expression has prognostic relevance in AML, we performed multivariate analysis with several factors influencing disease outcome. We did not identify an association between DNAM-1 expression and age, primary/secondary AML, risk profile according to the NCCN guideline and NPM1 and CEBPA mutations. Likewise, no significant differences in DNAM-1 expression between FAB subtypes with unfavorable  and favorable (M1-M5) prognosis were detected46. However, decreased DNAM-1 expression was associated with cases with myelodysplasia related changes when compared to other subtypes.Various activating immune receptors (e.g. Fc-receptors) are expressed on AML cellsWhen we employed ROC analysis and separated AML patients in two groups according to a defined cut-off value of 31.3% of DNAM-1 positive cells, a significant correlation of high DNAM-1 surface levels with better OS was observed. Notably, high DNAM-1 expression was also associated with improved OS and PFS in patients after HSCT.47. Attenuated expression of DNAM-1 on NK cells or DNAM-1 ligands on AML cells was suggested as an immune escape mechanism48. In agreement with this, higher expression of DNAM-1 on NK cells was associated with increased survival in AML patients49. The data presented here show that elevated DNAM-1 expression on AML cells themselves was also associated with improved survival of AML patients. So far, it remains unclear to which extent improved survival of individuals in the DNAM-1high group can in fact be attributed to engagement of the signaling axis or is rather an epiphenomenon of the association with more differentiated status of AML cells. This is even more since, as stated above, DNAM-1 expression was found to be associated with a CD34-negative blast phenotype and survival of patients negatively correlates with the amount of CD34-positive leukemic cells50.The role of NK cell-expressed DNAM-1 in recognition and killing of AML cells is well established. Interaction of DNAM-1 with its ligands CD112 and CD155 on AML cells is required for NK cell activation and cytotoxicityTo conclude, we here provide first evidence that DNAM-1 is functionally expressed on AML cells and expression correlates with survival of the patients. Based on the data reported, confirmatory studies in larger cohorts are warranted and hold promise to establish DNAM-1 expression as a prognostic marker for risk stratification and ultimately to improve outcome for AML patients.Supplementary Information 1.


Fagopyrum dibotrys (F. dibotrys) (D.Don) H.Hara is a well-known edible herbal medicine in Asian countries. It has been widely used for the treatment of lung diseases, swelling, etc., and is also an important part of many Chinese medicine prescriptions. At present, more than 100 compounds have been isolated and identified from F. dibotrys, and these compounds can be primarily divided into flavonoids, phenols, terpenes, steroids, and fatty acids. Flavonoids and phenolic compounds are considered to be the main active ingredients of F. dibotrys. Previous pharmacological studies have shown that F. dibotrys possesses anti-inflammatory, anti-cancer, anti-oxidant, anti-bacterial, and anti-diabetic activities. Additional studies on functional genes have led to a better understanding of the metabolic pathways and regulatory factors related with the flavonoid active ingredients in F. dibotrys. In this paper, we systemically reviewed the research advances on the phytochemistry and pharmacology of F. dibotrys, as well as the functional genes related to the synthesis of active ingredients, aiming to promote the development and utilization of F. dibotrys. Fagopyrum dibotrys (F. dibotrys) (D.Don) H.Hara, also called Fagopyrum acutatum (Lehm.) Mansf. ex K.Hammer, is a perennial herb of the dicotyledonaceae family Polygonaceae [F. dibotrys is widely distributed across the North temperate zone and is mainly grown in China, India, Thailand, and Vietnam. China is documented to be the main distribution region of F. dibotrys, and wild F. dibotrys has been listed as a national second-level protected plant [F. dibotrys are common food product used to make side dishes, flour, and tea, and its stem and leaf are used as forage feed resources.gonaceae . F. diboed plant . It has ed plant . The graF. dibotrys is also used as a traditional Chinese medicine for a long history [F. dibotrys possesses the effects of clearing away heat and detoxification, expelling pus and removing blood stasis, and is recommended for diseases such as lung abscesses, measles pneumonia, swelling and pain in throat and dysmenorrhea, inflammation, and so on. It is also an important part of many famous traditional Chinese medicine prescriptions such as Wei Mai Ning Capsule and Ji Zhi Tang Jiang.In addition to the nutritional and economic values, the rhizome of  history , 4. AccoF. dibotrys have identified more than 100 compounds, mainly including flavonoids, phenolics, triterpenoids and tannins, supporting its potential to be developed as a health care product and functional food [F. dibotrys possesses anti-inflammatory, anti-cancer, anti-oxidant, anti-bacterial, and anti-diabetic activities. The main bioactive components related with these pharmacological activities have been investigated, as well as the underlying mechanisms. Besides, with the developments in molecular biology technology, research into the functional genes helps to elucidate the biosynthetic pathways and regulatory mechanisms of active ingredients in medicinal plants. Additional studies on functional genes have led to a better understanding of the metabolic pathways and regulatory factors related with the active ingredients in F. dibotrys.In the past decades, phytochemical researches on nal food . PharmacF. dibotrys, aiming to provide critical information for further research on F. dibotrys.This article reviews research progress regarding the chemical compositions, pharmacological activities, and functional genes of Fagopyrum dibotrys belongs to the genus Fagopyrum (family Polygonaceae) which comprises more than ten species of plants. Among the Fagopyrum species, Fagopyrum esculentum (F. esculentum) (common buckwheat), Fagopyrum tataricum (F. tataricum) (tartary buckwheat), and F. dibotrys (golden buckwheat) attract increasing attention owing to their long history of both edible and medicinal uses [Fagopyrum species share similarities in nutritional and medicinal values, whereas there are also many differences between them.nal uses . These tFagopyrum esculentum and F. tataricum are annual herbs with small white or pinkish flowers and edible triangular seeds, while the seeds of F. tataricum are smaller than that of F. esculentum. F. esculentum and F. tataricum are extensively produced and consumed as the main buckwheats worldwide with more than 4000 years cultivation [F. tataricum, the seed of F. esculentum has advantages of sweet taste, large size, and easy dehulling of seed coat. However, detailed comparison studies indicated that the nutritional value of F. tataricum is much higher than F. esculentum. F. tataricum is richer in nutrients and antioxidants, such as vitamin B and rutin, and has good medicinal potential [F. tataricum are much higher compared to F. esculentum [F. dibotrys is a perennial wild herb with the characteristics of a thick main root, whorls, and large achenes. Similarly, the seeds of F. dibotrys are also edible, and its stem and leaf are also documented to be good forage feed resources [tivation . Compareotential , 8. The culentum . In contesources . Its leaF. dibotrys has been well investigated, and the chloroplast DNA size is detected to be 159,320 bp. Compared with the chloroplast DNA of other buckwheat species, F. dibotrys was more conserved with several variation hotspots [F. tataricum and F. esculentum together with two wild type F. dibotrys and F. luojishanense. Their results indicated that F. dibotrys is more closely related to Fagopyrum tataricum. By determining the DNA sequences of rbcL and accd59 coding regions and their intergenic regions in multiple taxa of Fagopyrum, it was found that the evolution speed of accd59 coding region and intergenic region were about 5 times that of the rbcL coding region. F. dibotrys has multiple polyploids, and F. dibotrys and F. tataricum are closely related [Fagopyrum species are still not fully understood, and much more work are needed.The existing methods to distinguish the buckwheat varieties mainly include morphology, allozyme variability, restriction fragment length polymorphism of chloroplast DNA, rbcL-accD region nucleotide sequence of chloroplast DNA, and nucleotide sequences of the ITS region of rRNA gene, etc. , 10. Thehotspots  analyzed related . HoweverF. esculentum and F. tataricum, F. dibotrys is more famous for its ethnomedicinal uses. F. dibotrys is an important crude drug which has been widely recorded in a series of Chinese medicine books, such as New Compilation of Materia Medica, Supplements to Compendim of Materia Medica. In China, the rhizome F. dibotrys is used as a folk herbal medicine for the treatment of lung disease, swelling and pain in throat and dysmenorrhea, inflammation, dysentery, and so on. In the Chinese Pharmacopoeia, F. dibotrys has been recorded to possess effects of heat-clearing and detoxicating, abscess and stasis removing.Compared with Fagopyrum dibotrys is also an important part of many modern Chinese patent medicine formulations, such as Wei Mai Ning Capsule, Ji Zhi Tang Jiang, Fu Ping Jiao Nang, Jin Hua Ming Mu Wan, Chang Xin Jiao Nang, Jin Ci Can Jiu Zheng He Ji, etc.  is a very famous over-the-counter drug which is effective in expectorating and relieving cough, and Fu Ping Jiao Nang  is used in clinic for diseases such as pelvic inflammation and accessory inflammation.c. Table . ThereinF. dibotrys have been widely studied in the past decades. To date, a number of compounds with diverse structures have been isolated from F. dibotrys, primarily including flavonoids, phenolics, triterpenoids, tannins, steroids, fatty acid, etc. Essential volatile oils of F. dibotrys consist of aliphatic compounds, aromatic compounds, and sulfur-containing nitrogen compounds. Compounds that have been isolated and identified are listed in Table The phytochemicals distributed in F. dibotrys. The content of flavonoids in F. dibotrys is higher than that found in other buckwheat cultivars. To date, more than 30 flavonoids have been isolated from F. dibotrys, such as quercetin and rutin. In addition, catechins, (+)-catechin, and (−)-epicatechin, have also been isolated from F. dibotrys. These isolated flavonoids are listed in Table Flavonoids are naturally occurring polyphenolic compounds characterized by a carbon-based skeleton (C6–C3–C6)  and are F. dibotrys. The main phenolic compounds are derivatives, such as benzoic acid and 3,4-dihydroxy benzoic acid. In addition, phenolic compounds such as gallic acid, succinic acid, syringic acid, and ferulic acid can be used directly in various applications.Phenolics refer to nonpolymeric phytochemical compounds that contain at least one phenolic hydroxyl located on the same benzene ring . PhenoliF. dibotrys. Among them, 3,3′-di-O-galloyl-procyanidin B-2 and 3′-O-galloyl-procyanidin B-2 are rich in hydroxyl groups.Tannins contain polyphenols and are widely reported to scavenge free radicals and prevent oxidation . Seven tF. dibotrys: glutinone, glutinol, 3α,21β-dihydroxy-olean-12-ene, olean-12-ene- 3β,7β,15α,28-tetraol, and ursolic acid.According to previous reports, a total of five triterpenoids have been extracted from F. dibotrys, mainly including 3α-hydroxy-urs-12,15-dien, Hecogenin, β-sitosterol, N-butanol-β-d-furan methylglycoside, n-butyl-β-d-fructopyronoside, β-daucosterol, and daucosterol.Aside from the compounds mentioned above, several steroids have also been identified from F. dibotrys. Eugenol, α-terpineol, nonanal, (E)-3-Hexen-1-ol and ethyl cinnamate are the main components. In addition, different types of volatile oils derived from F. dibotrys have been shown to have anti-bacterial and anti-oxidant effects [Essential volatile oils consist of aliphatic compounds, aromatic compounds, terpenes , and sulfur-containing nitrogen compounds. To date, more than forty constituents have been isolated from  effects . More deF. dibotrys contains many other compounds, such as emodin, emodin-8-O-β-d-glucopyranoside, N-trans-coumaroyl tyramine, (3-methoxyphenyl)-2-piperidinemethanol, n-butyl-β-d-fructopyronoside and 5,5′-di-α-furaldehyde dimethyl ether. According to previous studies [F. dibotrys is also rich in protein, starch, fat, crude fiber, vitamins, and mineral elements. Table In addition to the above components,  studies , F. diboF. dibotrys has been reported to possess a variety of pharmacological activities, including anti-inflammatory, anti-cancer, anti-oxidant, anti-viral, anti-diabetic and anti-bacterial activities  levels, thereby inhibiting NF-κB p65 nuclear translocation and IκB phosphorylation and improving LPS-induced cell proliferation. At the same time, F. dibotrys extract can also reduce levels of pro-inflammatory cytokines and be used as a treatment for ulcerative colitis [F. dibotrys can down-regulate tumor necrosis factor-α (TNF-α), as well interleukin (IL)-6, IL-1β and IL-10. By reducing expression of these inflammatory factors, F. dibotrys can thereby prevent and reduce inflammatory symptoms and damage [F. dibotrys has been shown to have a therapeutic effect on irritable bowel syndrome by up-regulating expression of claudin-1, claudin and ZO-1 tight junction proteins in colon epithelial cells, thereby reducing intestinal inflammation and enhancing mucosal epithelial function [F. dibotrys can inhibit experimental arthritis and reduce inflammation [F. dibotrys extract was shown to inhibit swelling and weight loss, reduce plasma viscosity, and significantly reduce production of proinflammatory cytokines IL-1 and TNF-α in serum. Treatment with F. dibotrys has also been shown to exert a protective effect on the lung tissue of rats with Klebsiella pneumonia [Inflammation represents a defense mechanism of different organisms to various harmful stimuli . Ethanol colitis . In cased damage . In bothfunction . Shen etammation . In expeneumonia .F. dibotrys is primarily derived from the monomer compounds isolated from it. Previous in vitro and in vivo experiments have demonstrated that anti-inflammation is one of the main pharmacological effects of luteolin, and the main mechanism include regulation of transcription factors STAT3, NF-κB and AP-1 [F. dibotrys, has been proven to inhibit LPS-induced inflammation in human gingival fibroblasts by activating the Nrf2 signaling pathway [The anti-inflammatory activity of and AP-1 . Isorham pathway . Chrysoe pathway . Tricin  pathway . Hespere pathway . In the  pathway . Similar pathway . In para pathway . Procyan pathway . Procyan pathway .F. dibotrys has received more and more attention. Many studies have demonstrated that F. dibotrys and its components exhibit significant in vitro and in vivo inhibitory effects in various cancer types. F. dibotrys extract treatment could inhibit the growth of various cancer cells including lung cancer (H460), liver cancer (HepG2), leukocytes (K562), colon cancer (HCT116) and bone cancer (U2OS) [F. dibotrys extract administration can inhibit the transplant ability of S180 sarcoma, Lewis lung cancer and U14 cervical cancer in mice models [F. dibotrys extract with more than 50% polyphenol content, it can down-regulate expression of matrix metalloproteinase (MMP)-9 and significantly inhibit the progression of Lewis lung cancer in mice [In the past decades, the anti-cancer activity of r (U2OS) . F. diboe models , 34. Fr4 in mice .F. dibotrys extract in combination with other drugs have been documented to increase the efficacy and reduce the toxicity of radiotherapy and chemotherapy. Wei Mai Ning Capsule which contains extract of F. dibotrys as the main raw material has been approved for clinical cancer therapy. Combination of extracts from Rosa roxburghii Tratt and F. dibotrys significantly promoted apoptosis and inhibited growth of human esophageal squamous cell carcinoma CaEs-17, human gastric cancer SGC-7901 and lung cancer A549 cells [F. dibotrys extract has also been shown to have a synergistic effect.Cancer prevention and treatment using combination therapy with Chinese medicines have been proposed as an effective strategy in clinic during the past years , 37. Par49 cells . The comF. dibotrys have been investigated, as well as the underlying mechanisms. β-sitosterol (β-SITO), a compound found in F. dibotrys, has been demonstrated to decrease the viability of breast cancer MCF-7 cells, and immunofluorescence analysis confirmed the tubulin-targeted anticancer potential of β-SITO [F. dibotrys and previous studies have shown that ursolic acid can block the G1/G2 cell cycle and induce breast cancer cell apoptosis [The main components responsible for the anti-cancer activity of f β-SITO . (−)-Epif β-SITO . Murexinf β-SITO . Rhamnetf β-SITO . Isorhamf β-SITO . Genkwanf β-SITO . Many stf β-SITO . In addif β-SITO . Ursolicpoptosis . Moreovepoptosis .Fagopyrum dibotrys contains a high concentration of polyphenols and flavonoids, which possess the ability to scavenge free radicals and superoxide anions. It has been documented to exhibit strong anti-oxidant capacity [F. dibotrys extract exhibited stronger anti-oxidant activity, and the ability of 6-O-galloyl-d-glucose to scavenge free radicals is even higher than that of vitamin C. Extracts of different parts of F. dibotrys have different anti-oxidant properties, with leaf extract showing the strongest anti-oxidant properties [capacity . This ancapacity , 50. Manoperties .2O2-induced 8-oxo-7,8-dihydro-2 V-deoxyguanosine in human leukemia cell line HL-60. However, at high concentrations, it will increase the formation of 8-oxodG in HL-60 cells. Experiments with calf thymus DNA showed that procyanidin B2 reduced the M4PO-OH signal of H2O2 and Fe(II), but enhanced H2O2 and Cu(II) signals and induced DNA damage. Therefore, procyanidin B2 may interact with H2O2 and metal ions to exert both anti-oxidant and pro-oxidant effects [Hesperidin has a variety of biological activities, especially anti-inflammatory effects which is manifested in two main aspects: scavenging free radical activity and increasing the defense ability of anti-oxidant cells through the ERK/Nf2 signaling pathway . Ferulic effects .F. dibotrys has been proved to exhibit obvious anti-bacterial effect against Streptococcus haemolyticus B and pneumococcus in vitro. In vivo, the extract had an obvious protective effect on mice infected by pneumococcus. Based on the previous reports, the main anti-bacterial compounds are believed to be the phenolic acids, flavonoids and flavanol compounds [F. dibotrys and levofloxacin can improve the treatment efficacy of acute bacterial dysentery [F. dibotrys can be used to treat the lung tissue damage caused by Klebsiella pneumoniae by reducing expression of TNF-α, ICAM-1, NF-κB p65 and MIP-2 [F. dibotrys was formulated into ultrafine powder and used at high, medium and low doses in mice infected with Salmonella. The results demonstrated that the ultrafine F. dibotrys powder had a protective effect on mice infected with Salmonella and suggested a dose-activity relationship [The ethyl acetate extract of ompounds . The comysentery . Studiesnd MIP-2 . In anottionship .Streptococcus pneumoniae. β-SITO directly attacks the pneumolysin, the main virulence factor of pneumococcus, through two action sites (Thr459 and Leu460) to prevent cell lysis [Salmonella typhimurium [Haemophilus parasui by interacting with the cell membrane or proteins on the cell wall to change the conformation of membrane proteins [Due to the abuse of antibiotics, antibiotic resistance has become a major threat to public health . Discovell lysis . In studproteins . Bacteriproteins .F. dibotrys. Zhao et al. evaluated the anti-viral potential of F. dibotrys using both the hemagglutination test and a cell culture method, their results indicated F. dibotrys has anti-viral activity in vitro, and this activity is dependent on the dose of F. dibotrys flavonoids [Since the treatment of viral influenza with chemical drugs is prone to induce adverse reactions, such as drug resistance , more anavonoids .African swine fever virus (ASFV) is a serious threat to pig breeding due to the lack of effective treatment measures. In vitro experiments have shown that genkwanin can reduce protein levels and DNA synthesis in both early and late stages of ASFV, in addition to inhibiting the ASFV Ba71V strain in Vero cells . Tricin F. dibotrys extract is rich in amino acids and vitamins which can scavenge free radicals, reducing damage to the body and achieve anti-diabetic effects. Treatment with 50–200 mg/kg F. dibotrys flavonoids is effective in type 2 diabetic mice. In T2DM mice, this treatment can reduce blood glucose and insulin levels, increase body weight and regulate blood lipid metabolism and oxidative stress [P-coumaric administration led to a significant reduction in the levels of glucose and glycosylated hemoglobin, as well as induced significant increases in insulin and body weight. Additional anti-diabetic mechanisms of P-coumaric acid include reduced levels of TNF-α and increased levels of adipocytokines and PPARγ [Previous studies indicated that reactive oxygen radicals and their oxidation reactions can damage islet β cells, leading to diabetes . F. diboe stress . In vitre stress . Procyane stress . Moreovend PPARγ .F. dibotrys also has several other activities. F. dibotrys contains a lot of polyphenolic compounds and has obvious anti-tussive and expectorant effects [F. dibotrys has a therapeutic effect on Alzheimer’s disease (AD) [F. dibotrys flakes to rats, the collected serum can inhibit the contraction of guinea pig ileum in vitro [F. dibotrys has an anti-mutagenic effect on mutations of TA98 and TA100 strains induced by orthodoxin and methanesulfonate [In addition to the above pharmacological activities,  effects . It alsoase (AD) . After iin vitro . In addiulfonate .Fagopyrum dibotrys extract has therapeutic potential for neurodegenerative diseases, and vitexin is suggested to be one of the main active compounds. Vitexin can increase neuroprotective factors and counteract the induction of neurodegeneration. Ferulic acid is also considered to be a promising treatment for AD [t for AD . Ferulict for AD . The neut for AD . Procyant for AD .F. dibotrys have been studied, the genes related with the secondary metabolites of F. dibotrys have been discovered as well as the relevant metabolic pathways. Relative studies play an important role in research into the active ingredients of F. dibotrys. F. dibotrys contains many flavonoids, and its biological activity and synthetic pathways have received extensive attention. At present, the research into functional genes of F. dibotrys is primarily focused on flavonoids.At present, research into medicinal plants primarily focuses on extraction and separation technology, effective ingredient analysis and assessment of pharmacological activity . With inF. dibotrys for the first time using homologous cloning technology. Bioinformatics showed that the protein encoded by the FdANS gene was homologous to the ANS genes of other medicinal plants. The expression levels of FdANS in different tissues were analyzed; the results showed that the level of FdANS was flower > leaf > stem > root, but the amount of anthocyanin was flower > leaf > stem > root. The amount of penicillin is correlated [F. dibotrys contains a lot of procyanidins (PAs). A radiation-induced mutant (RM_R) obtained by Chen et al. [Anthocyanins are flavonoids, and the anthocyanin synthase (ANS) gene is critical to the anthocyanin synthesis pathway in golden buckwheat. The anthocyanin synthase (FdANS) gene was obtained from rrelated . The rhin et al.  containeF. dibotrys. The DFR gene in the F. dibotrys genome is a small family of 1 to 2 genes. FdDFR1 is a single copy gene and is homologous to DFR genes of other medicinal plants [F. dibotrys by PCR and RACE. By using real-time fluorescence quantitative PCR technology, researchers found that the FdLAR gene was related with the accumulation of secondary metabolites of buckwheat flavonoids [F. dibotrys flavonoid synthesis pathway [F. dibotrys by using homologous cloning technology. Bioinformatic analysis showed that the protein encoded by the FdCHI gene was homologous to the CHI genes of other medicinal plants, and FdCHI was expressed in different tissues of F. dibotrys. The results showed that expression of FdCHI was flower > root > leaf > stem, while total flavonoid content was flower > leaf > stem > root. This suggests that expression of FdCHI is related to the total flavonoid content in flowers, leaves and stems, but is less relevant in the root [Dihydroflavonol 4-reductase (DFR) is a key enzyme for procyanidin synthesis. Using the RACE method combined with cDNA library screening, the DFR (FdDFR) gene was cloned from l plants . A mixtul plants . The leuavonoids . In addi pathway . The cDNthe root .F. dibotrys. Rutin belongs to the flavonoid family, and its synthesis is inseparable from that of phenylalanine ammonia lyase (PAL). Through homologous cloning and RT-PCR technology, the DNA sequence of the F. dibotrys PAL gene was measured to be 2583 bp. The cDNA sequence contained an open reading frame of 2169 bp, encoding 722 amino acids. After synthesis, the acid was converted to cinnamic acid [F. dibotrys was determined [Rutin is another secondary metabolite in mic acid . The spemic acid . Flavanomic acid , 92. Usitermined .F. dibotrys flavonoid synthesis pathway. Some studies have used RACE combined with a cDNA library screening method to clone the transcription factor gene MYBP1 (FdMYBP1) from F. dibotrys. Through southern hybridization and bioinformatics analysis, researchers revealed that FdMYBP1 gene exists in one to two copies, equal to MYB based on structural characteristics of the source gene, it is believed that FdMYBP1 plays a vital role in flavonoid metabolism [Many transcription factors also play important roles in the tabolism .Fagopyrum dibotrys is a famous edible herbal medicine widely used in traditional Chinese medicine. In China, F. dibotrys has been used for a long time to treat diseases such as pneumonia and tonsil swelling. As a traditional Chinese medicine, F. dibotrys is an important component of many Chinese medicine formulas, including Wei Mai Ning Capsule, Ji Zhi Tang Jiang, and Jin Hua Ming Mu Wan. To date, more than 70 chemical components have been isolated from F. dibotrys, together with nearly 50 components from the volatile oil. However, investigation on the whole chemical components of F. dibotrys is far from finish up completely. With the development of modern science and technologies in rapid and efficient separation and identification of complex chemical compounds from natural products [F. dibotrys using technologies such as multi-dimensional liquid phase chromatography combined with mass spectrometry. Better understanding on phytochemistry will undoubtedly promote the further investigation of golden buckwheat.products , it is eF. dibotrys has characterized its multiple effects which partially support its traditional uses. For example, a series of pharmacological studies focused on the anti-inflammatory activity of F. dibotrys and have proved its therapeutic potential in lung abscesses, irritable bowel syndrome, etc. The extracts of F. dibotrys and isolated compounds including hecogenin, luteolin, isorhamnetin and chrysoeriol have been demonstrated to suppress secretion of inflammatory cytokines and inhibit related pathways in relevant in vitro and in vivo models. Besides, according to the ethnomedicinal uses in China, Jin Ci Can Jiu Zheng He Ji, which is composed of Rosa roxburghii, Sophora flavescens and F. dibotrys, has been clinically used in adjuvant therapy for radiotherapy and chemotherapy. Accordingly, modern pharmacological studies demonstrated that the extracts and monomers of F. dibotrys exhibited anti-cancer activities in various cancer types in vitro and in vivo. Furthermore, Wei Mai Ning Capsule has been clinical used to increase the efficacy and reduce the toxicity of radiotherapy and chemotherapy. However, the main components responsible for the anti-cancer potential of F. dibotrys are not fully clarified, as well as the underlying molecular mechanisms. With the widespread application of targeted therapy and immunotherapy in clinical cancer treatment, the combination effects of F. dibotrys-related products with relative therapeutic strategies also worth further evaluation.Previous research on the pharmacological activity of F. dibotrys have attracted most of the attention, while investigation on the pharmacological activities that supporting the folk medicinal usage of F. dibotrys are still insufficient. For example, F. dibotrys has been documented as an important Chinese medicine for its therapeutic effect against pulmonary abscess and pneumonia. However, the potential mechanisms and targets are still unclear. With the development of traditional Chinese medicine and the strengthening of the world’s understanding of traditional Chinese medicine, research on the anti-inflammation, anti-viral and immune-promoting effects of F. dibotrys should be synchronously promoted, and much more work is still needed. Previously, F. dibotrys extracts or monomers are mostly used in the in vivo and in vitro pharmacological studies. Studies have proved that F. dibotrys has a certain effect on pneumonia, enteritis, diabetes, and other diseases. However, in some of the pharmacological studies, the in vivo experimental dose is much too high, and some in vitro experimental administration time is very long, whether these experimental designs are reasonable should be taken into consideration in the future studies.Previously, the anti-cancer and anti-oxidant activities of F. dibotrys is mainly concentrated on flavonoids. In subsequent endeavors, research into other secondary metabolites including the polyphenols should be strengthened in order to further clarify the synthetic pathways and regulatory mechanisms of the effective ingredients of F. dibotrys.With developments in biochemistry, people are paying more and more attention to the secondary metabolites when researching medicinal plants. It has been documented that the contents of secondary metabolites in medicinal plants are related with the expression of different functional genes. Therefore, it is critical to start from functional genes to elucidate the biosynthetic pathways and regulatory mechanisms of active ingredients involved in the regulation of secondary metabolites and structure-encoding genes of key enzyme systems . PreviouF. dibotrys has also received significant attention because of its high nutritional and medicinal value. F. dibotrys seeds are often used as coarse grains and can be made into tea. F. dibotrys leaves can be eaten directly as vegetables [F. dibotrys can be processed into highly nutritious feed or feed additives, and green straw can be used as pasture. Modern food processing technology and equipment may help further development of F. dibotrys related healthy products. More importantly, protection of the wild resources and development of cultivation techniques and genetic analysis will help better development and utilization of this medicinal economic plant.In addition to the aforementioned phytochemical and pharmacological studies, getables . F. diboF. dibotrys has attracted increasing attention in the past decades. To date, more than 100 compounds have been isolated and identified from F. dibotrys, mainly including flavonoids, phenols, steroids, and fatty acids, etc. Pharmacological studies have shown that F. dibotrys possesses anti-inflammatory, anti-cancer, anti-oxidant, anti-bacterial and anti-diabetic activities, which partially support the ethnomedicinal uses of F. dibotrys. Flavonoids and phenolic compounds are considered to be the main active ingredients of F. dibotrys. Additional studies on functional genes have led to a better understanding on the metabolic pathways and regulatory factors related to flavonoid active ingredients in F. dibotrys. Our study provides a comprehensive review regarding the research advances on the phytochemistry and pharmacology of F. dibotrys, as well as the functional genes, aiming to promote further research on F. dibotrys.As a famous edible herbal medicine widely used in traditional Chinese medicine,